Genetic engineering salt tolerance in crop plants

ABSTRACT

The invention is isolated nucleic acid molecules encoding Na + /H +  exchanger polypeptides with at least 95% homology to that of  Arabidopsis thaliana  for extrusion of monovalent cations from the cytosol of cells to provide the cell with increased salt tolerance. Crop species transformed with the nucleic acid molecule are capable of surviving in soil with high salt levels that would normally inhibit growth of the crop species.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/271,584, filed Mar. 18, 1999, now U.S. Pat No. 7,041,875,which claims priority to U.S. patent application Ser. Nos. 60/116,111,filed Jan. 15, 1999, and 60/078,474, filed Mar. 18, 1998, each of whichis hereby incorporated in its entirety.

BACKGROUND OF THE INVENTION

Environmental stress due to salinity is one of the most serious factorslimiting the productivity of agricultural crops, which are predominantlysensitive to the presence of high concentrations of salts in the soil.Large terrestrial areas of the world are affected by levels of saltinimical to plant growth. It is estimated that 35-45% of the 279 millionhectares of land under irrigation is presently affected by salinity.This is exclusive of the regions classified as arid and desert lands,(which comprises 25% of the total land of our planet). Salinity has beenan important factor in human history and in the life spans ofagricultural systems. Salt impinging on agricultural soils has createdinstability and has frequently destroyed ancient and recent agrariansocieties. The Sumerian culture faded as a power in the ancient worlddue to salt accumulation in the valleys of the Euphrates and Tigrisrivers. Large areas of the Indian subcontinent have been renderedunproductive through salt accumulation and poor irrigation practices. Inthis century, other areas, including vast regions of Australia, Europe,southwest USA, the Canadian prairies and others have seen considerabledeclines in crop productivity.

Although there is engineering technology available to combat thisproblem, though drainage and supply of high quality water, thesemeasures are extremely costly. In most of the cases, due to theincreased need for extensive agriculture, neither improved irrigationefficiency nor the installation of drainage systems is applicable.Moreover, in the arid and semi-arid regions of the world waterevaporation exceeds precipitation. These soils are inherently high insalt and require vast amounts of irrigation to become productive. Sinceirrigation water contains dissolved salts and minerals, an applicationof water is also an application of salt that compounds the salinityproblem.

Increasing emphasis is being given to modify plants to fit therestrictive growing conditions imposed by salinity. If economicallyimportant crops could be manipulated and made salt resistant, this landcould be farmed resulting in greater sales of seed and greater yield ofuseful crops. Conventional breeding for salt tolerance has beenattempted for a long time. These breeding practices have been basedmainly on the following strategies: a) the use of wide crosses betweencrop plants and their more salt-tolerant wild relatives (1), b)screening and selecting for variation within a particular phenotype (2),c) designing new phenotypes through recurrent selection (3). The lack ofsuccess in generating tolerant varieties (given the low number ofvarieties released and their limited salt tolerance) (4) would suggestthat conventional breeding practices are not enough and that in order tosucceed a breeding program should include the engineering of transgeniccrops (5).

Several biochemical pathways associated with stress tolerance have beencharacterized in different plants and a few of the genes involved inthese processes have been identified and in some cases the possible roleof proteins has been investigated in transgenic/overexpressionexperiments. Several compatible solutes have been proposed to play arole in osmoregulation under stress. Such compatible solutes, includingcarbohydrates (6), amino acids (7) and quaternary N-compounds (8) havebeen shown to increase osmoregulation under stress. Also, proteins thatare normally expressed during seed maturation (LEAs, Late EmbriogenesisAbundant proteins) have been suggested to play a role in water retentionand in the protection of other proteins during stress. Theoverexpression of LEA in rice provided a moderate benefit to the plantsduring water stress (9,10). A single gene (sod2) coding for a Na⁺/H⁺antiport has been shown to confer sodium tolerance in fission yeast(11,12), although the role of this plasma membrane-bound protein appearsto be only limited to yeast. One of the main disadvantages of using thisgene for transformation of plants is associated with the typicalproblems encountered in heterologous gene expression, i.e. incorrectfolding of the gene product, targeting of the protein to the targetmembrane and regulation of the protein function.

Plants that tolerate and grow in saline environments have highintracellular salt levels. A major component of the osmotic adjustmentin these cells is accomplished by ion uptake. The utilization ofinorganic ions for osmotic adjustment suggests that salt-tolerant plantsmust be able to tolerate high levels of salts within their cells.However, enzymes extracted from these plants show high sensitivity tosalt. The sensitivity of the cytosolic enzymes to salt would suggestthat the maintenance of low cytosolic sodium concentration, either bycompartmentation in cell organelles or by exclusion through the plasmamembrane, must be necessary if the enzymes in the cell are to beprotected from the inimical effects of salt.

Plant cells are structurally well suited to the compartmentation ofions. Large membrane-bound vacuoles are the site for a considerableamount of sequestration of ions and other osmotically active substances.A comparison of ion distribution in cells and tissues of various plantspecies indicates that a primary characteristic of salt tolerant plantsis their ability to exclude sodium out of the cell and to take up sodiumand to sequester it in the cell vacuoles. Transport mechanisms couldactively move ions into the vacuole, removing the potentially harmfulions from the cytosol. These ions, in turn, could act as an osmoticumwithin the vacuole, which would then be responsible for maintainingwater flow into the cell. Thus, at the cellular level both specifictransport systems for sodium accumulation in the vacuole and sodiumextrusion out of the cell are correlated with salt tolerance.

SUMMARY OF THE INVENTION

We have isolated the first such system of intracellular salt management.We identified the presence of a functional vacuolar Na⁺/H⁺ antiport inthe vacuolar membrane of higher plants (13,14,15,16,17,18).

We have demonstrated the Na⁺/H⁺ antiport function in isolated tonoplastmembranes and in intact vesicles and we showed that the activity ofantiport molecules was salt dependent. Neither a protein sequence nor agene encoding the antiport were identified in previously published work.We have now identified nucleic acid molecules coding for plant Na⁺/H⁺antiports, the nucleic acid molecules and polypeptides produced by thenucleic acid molecules being the subject of the present invention. Thesepolypeptides are useful for the extrusion of sodium ions from thecytosol, either through the accumulation of sodium ions into thevacuoles or into the extracellular space, thus providing the mostimportant trait for salt tolerance in plants. These nucleic acidmolecules, preferably genes, are useful for the engineering of salttolerant plants by transformation of salt-sensitive crops overexpressingone or more of these nucleic acid molecules under the control ofconstitutively active promoters or under the control ofconditionally-induced promoters. Agrobacterium tumefaciens-mediatedtransformation or particle-bombardment-mediated transformation areuseful for depending upon the plant species.

The invention includes an isolated nucleic acid molecule encoding a PNHXtransporter polypeptide, or a fragment of a polypeptide having Na⁺/H⁺transporter activity and capable of increasing salt tolerance in a cell.

The invention also relates to an isolated nucleic acid molecule encodinga THX transporter polypeptide, PNHX transporter polypeptide, or afragment of a polypeptide having Na⁺/H⁺ transporter activity and capableof increasing salt tolerance in a cell, comprising a nucleic acidmolecule selected from the group consisting of:

-   -   (a) a nucleic acid molecule that hybridizes to all or part of a        nucleic molecule in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:17, SEQ        ID NO:19, or a complement thereof under moderate or high        stringency hybridization conditions, wherein the nucleic acid        molecule encodes a TNHX transporter polypeptide, a PNHX        transporter polypeptide or a polypeptide having Na⁺/H⁺        transporter activity and capable of increasing salt tolerance in        a cell;    -   (b) a nucleic acid molecule degenerate with respect to (a),        wherein the nucleic molecule encodes a TNHX transporter        polypeptide, a PNHX transporter polypeptide or a polypeptide        having Na⁺/H⁺ transporter activity and capable of increasing        salt tolerance in a cell.

The hybridization conditions preferably comprise moderate (also calledintermediate) or high stringency conditions selected from the conditionsin Table 4.

The invention also includes an isolated nucleic acid molecule encoding aTHX transporter polypeptide or a PNHX transporter polypeptide, or afragment of a polypeptide having Na⁺/H⁺ transporter activity and capableof increasing salt tolerances in a cell, comprising a nucleic acidmolecule selected from the group consisting of:

-   (a) the nucleic acid molecule of the coding strand shown in SEQ ID    NO:1, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:19 or a complement    thereof;-   (b) a nucleic acid molecule encoding the same amino acid sequence as    a nucleotide sequence of (a); and-   (c) a nucleic acid molecule having at least 17% identity with the    nucleotide sequence of (a) and which encodes a THX transporter    polypeptide or the PNHX transporter polypeptide or a polypeptide    having Na⁺/H⁺ transporter activity.

The THX transporter polypeptide or the PNHX transporter polypeptidepreferably comprises an AtNHX transporter polypeptide having Na⁺/H⁺transporter activity and capable of increasing salt tolerance in a cell.The nucleic acid molecule may comprise all or part of a nucleotidesequence in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:17 or SEQ ID NO:19 (orthe coding region therof).

The invention also includes an AtNHX nucleic acid molecule isolated fromArabidopsis thaliana, or a fragment thereof encoding a transporterpolypeptide having Na⁺/H⁺ transporter activity and capable of increasingsalt tolerance in a cell.

Another aspect of the invention relates to a recombinant nucleic acidmolecule comprising a nucleic acid molecule and a constitutive promotersequence or an inducible promoter sequence, operatively linked so thatthe promoter enhances transcription of the nucleic acid molecule in ahost cell.

The nucleic acid molecule preferably comprises genomic DNA, cDNA or RNA.In another aspect, the nucleic acid molecule is chemically synthesized.The nucleic acid molecule is preferably isolated from Arabidopsisthaliana.

The nucleic acid molecule preferably encodes a TNHX transporterpolypeptide or PNHX transporter polypeptide that is capable of extrudingmonovalent cations out of the cytosol of a cell to provide the cell withincreased salt tolerance, wherein the monovalent cations are selectedfrom at least one of the group consisting of sodium, lithium andpotassium. The cell preferably comprises a plant cell. The monovalentcations are preferably extruded into a vacuole or into the extracellularspace.

The invention also includes an isolated nucleic acid molecule comprisinga nucleic acid molecule selected from the group consisting of 8 to 10nucleotides of the nucleic acid molecules described above, 11 to 25nucleotides of the nucleic acid molecules described above, and 26 to 50nucleotides of the nucleic acid molecules described above.

The invention also includes an isolated oligonucleotide comprising atleast about 10 nucleotides from a sequence selected from the groupconsisting of 5′-GCCATGTTGGATTCTCTAGTGTCG-3 (SEQ ID NO:11),5′-CCGAATTCTCAAAGCTTTTCTTCCACG-3′ (SEQ ID NO:12),5′-CGGAATTCACAGAAAAACACAGTGAGGAT-3′ (SEQ ID NO:13),5′-GCCATGTTGGATTCTCTAGTGTCG-3 (SEQ ID NO:14),CCGAATTCTCAAAGCTTTTCTTCCACG-3′ (SEQ ID NO:15),5′-CGGAATTCACAGAAAAACACAGTGAGGAT-3′ (SEQ ID NO:16) or anotheroligonucleotide described in this application.

Another aspect of the invention relates to a vector comprising a nucleicacid molecule of the invention. The vector preferably comprises apromoter selected from the group consisting of a super promoter, a 35Spromoter of cauliflower mosaic virus, a drought-inducible promoter, anABA-inducible promoter, a heat shock-inducible promoter, asalt-inducible promoter, a copper-inducible promoter, asteroid-inducible promoter and a tissue-specific promoter.

The invention also includes a host cell comprising a recombinant nucleicacid molecule of the invention, or progeny of the host cell.

The host cell is preferably selected from the group consisting of afungal cell, a yeast cell, a bacterial cell, a microorganism cell and aplant cell. The plant, a plant part, a seed, a plant cell or progenythereof preferably comprises the recombinant nucleic acid molecule ofthe invention. The plant part preferably comprises all or part of aleaf, a flower, a stem, a root or a tuber. The plant, plant part, seedor plant cell is preferably of a species selected from the groupconsisting of potato, tomato, brassica, cotton, sunflower, strawberries,spinach, lettuce, rice, soybean, corn, wheat, rye, barley, atriplex,sorgum, alfalfa, salicornia and the plant species or types in Table 5.

The plant, plant part, seed or plant cell preferably comprises a dicotplant or a monocot plant.

The invention also relates to a method for producing a recombinant hostcell capable of expressing the nucleic acid molecule of the invention,the method comprising introducing into the host cell a vector of theinvention. The invention also includes a method of producing agenetically transformed plant which expresses TNHX or PNHX transporterpolypeptide, comprising regenerating a genetically transformed plantfrom a plant cell, seed or plant part of the invention. In one method,the genome of the host cell also includes a functional TNHX or PNHXgene. In another method, the genome of the host cell does not include afunctional TNHX or PNHX gene. The invention also includes a transgenicplant produced according to a method of the invention.

Another aspect of the invention relates to a method for expressing aTNHX or PNHX transporter polypeptide in the host cell of the invention,a the plant, plant part, seed or plant cell of the invention, the methodcomprising culturing the host cell under conditions suitable for geneexpression. A method for producing a transgenic plant that expresseselevated levels of PNHX transporter polypeptide relative to anon-transgenic plant, comprising transforming a plant with the vector ofthe invention. The invention also relates to an isolated polypeptideencoded by and/or produced from a nucleic acid molecule of theinvention, or the vector of the invention.

The invention also relates to an isolated PNHX transporter polypeptideor a fragment thereof having Na⁺/H⁺ transporter activity and capable ofincreasing salt tolerance in a cell. The polypeptide of the inventionpreferably comprises an AtNHX transporter polypeptide. The polypeptideof the invention preferably comprises all or part of an amino acidsequence in SEQ ID NOS: 2, 4, 6, 8, 18, or 20 (FIG. 1). The inventionalso includes a polypeptide fragment of the AtNHX transporterpolypeptide of the invention, or a peptide mimetic of the AtNHXtransporter polypeptide, having Na⁺/H⁺ transporter activity and capableof increasing salt tolerance in a cell. The polypeptide fragment of theinvention, preferably consists of at least 20 amino acids, whichfragment has Na⁺/H⁺ transporter activity and is capable of increasingsalt tolerance in a cell. The fragment or peptide mimetic of theinvention is preferably capable of being bound by an antibody to apolypeptide of the invention. In one embodiment, the polypeptide of theinvention is recombinantly produced.

The invention also includes an isolated and purified transporterpolypeptide comprising the amino acid sequence of a TNHX transporterpolypeptide or a PNHX transporter polypeptide, wherein the transporterpolypeptide is encoded by a nucleic acid molecule that hybridizes undermoderate or stringent conditions to a nucleic acid molecule in SEQ IDNOS: 1, 3, 5, 7, 17, or 19 (FIG. 1), a degenerate form thereof or acomplement. The invention also includes a polypeptide comprising asequence having greater than 28% sequence identity to a polypeptide ofthe invention (preferably a polypeptide in FIG. 1, such as SEQ ID NOS:2, 4, 6, 8, 18, or 20).

The polypeptide of the invention, preferably comprises a Na⁺/H⁺transporter polypeptide. The polypeptide is preferably isolated fromArabidopsis thaliana.

The invention also includes an isolated nucleic acid molecule encodingpolypeptide of the invention (preferably a polypeptide in FIG. 1: SEQ IDNOS: 2, 4, 6, 8, 18, or 20).

Another aspect of the invention relates to an antibody directed againsta polypeptide of the invention. The antibody of the invention,preferably comprises a monoclonal antibody or a polyclonal antibody.

The invention also relates to an isolated nucleic acid molecule encodinga TNHX transporter polypeptide or a PNHX transporter polypeptide, or afragment of a polypeptide having Na⁺/H⁺ transporter activity and capableof increasing salt tolerance in a cell, comprising a nucleic acidmolecule selected from the group consisting of:

-   -   (a) a nucleic acid molecule that hybridizes to all or part of a        nucleic molecule in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or to        a nucleic acid molecule comprising about nucleotides 1-1487 of        SEQ ID NO:9, or a complement thereof under moderate or high        stringency hybridization conditions, wherein the nucleic acid        molecule encodes a TNHX transporter polypeptide, a PNHX        transporter polypeptide or a polypeptide having Na⁺/H⁺        transporter activity and capable of increasing salt tolerance in        a cell;    -   (b) a nucleic acid molecule degenerate with respect to (a),        wherein the nucleic molecule encodes a TNHX polypeptide, a PNHX        polypeptide or a polypeptide having Na⁺/H⁺ transporter activity        and capable of increasing salt tolerance in a cell.    -   (c) the nucleic acid molecule of the coding strand shown in SEQ        ID NO:5, SEQ ID NO:7, SEQ ID NO:9, nucleotides 1-1487 of SEQ ID        NO:9, or an isolated nucleic acid molecule including about 1614        nucleic acids including SEQ ID NO:5, SEQ ID NO:7, nucleotides 1        to 1487 of the nucleic acid molecule in SEQ ID NO:9 or the        complement thereof;    -   (d) a nucleic acid molecule encoding the same amino acid        sequence as a nucleotide sequence of (c); and    -   (e) a nucleic acid molecule having at least 17% sequence        identity with the nucleotide sequence of (c) and which encodes a        TNHX transporter polypeptide, a PNHX transporter polypeptide or        a polypeptide having Na⁺/H⁺ transporter activity and capable of        increasing salt tolerance in a cell.

The invention also includes a polypeptide produced from a nucleic acidmolecule of the invention. The invention includes a polypeptidecomprising (a) the amino acid sequence in SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10; (b) amino acids 1 to 496 of SEQ ID NO:10; and (c) a sequencehaving greater than 28% homology to the polypeptide in (a) or (b). Theinvention includes a polypeptide comprising a Na⁺/H⁺ transporterpolypeptide capable of increasing salt tolerance in a cell. Theinvention also includes a DNA molecule encoding the polypeptides of theinvention.

The invention relates to a method of producing a genetically transformedplant which expresses or overexpresses a TNHX transporter polypeptide, aPNHX transporter polypeptide or a polypeptide having Na⁺/H⁺ transporteractivity and capable of increasing salt tolerance in a cell and whereinthe plant has increased salt tolerance, comprising:

-   a) cloning or synthesizing a TNHX nucleic acid molecule, a PNHX    nucleic acid molecule or a nucleic acid molecule which codes for a    Na⁺/H⁺ transporter polypeptide, wherein the polypeptide is capable    of providing salt tolerance to a plant;-   b) inserting the nucleic acid molecule in a vector so that the    nucleic acid molecule is operably linked to a promoter;-   c) inserting the vector into a plant cell or plant seed;-   d) regenerating the plant from the plant cell or plant seed, wherein    salt tolerance in the plant is increased compared to a wild type    plant.

The invention includes a transgenic plant produced according to a methodof the invention.

The nucleic acid molecules have several uses which will be discussed inmore detail below. The nucleic acid molecules and the polypeptides areused in a method for protecting a plant from the adverse affects of asaline environment by incorporating a nucleic acid molecule for salttolerance and/or the polypeptide of the invention into a plant. Thenucleic acid molecules of the invention are also useful for theidentification of homologous nucleic acid molecules from plant species,preferably salt tolerant species and genetically engineering salttolerant plants of agricultural and commercial interest.

The invention relates to isolated nucleic acid molecules encoding apolypeptide for extrusion of sodium ions from the cytosol of a cell toprovide the cell with salt tolerance. The nucleic acid moleculespreferably comprise the nucleotide sequence in FIG. 1( a) or (b). Thenucleic acid molecules may be DNA or RNA. The nucleic acid molecules maybe used to transform a cell selected from the group consisting of aplant cell, a yeast cell and a bacterial cell. The sodium ions areextruded into a vacuole or out of the cell. The nucleic acid moleculesencode a Na⁺/H⁺ exchanger polypeptide.

In a preferred embodiment, the nucleic acid molecules are isolated fromArabidopsis thaliana.

The invention includes an isolated nucleic acid molecule, comprising theDNA sequence in FIG. 1( a), (b), (c)(i), (c)(ii), (d) or (e). Theinvention also relates to an isolated nucleic acid molecule, comprisinga sequence having greater than 17% homology to the sequences of theinvention described in the preceding paragraphs.

In an alternate embodiment, the nucleic acid molecule consists of asequence selected from the group consisting of 8 to 10 nucleotides ofthe nucleic acid molecules of the invention, 11 to 25 nucleotides of thenucleic acid molecule and 26 to 50 nucleotides of the nucleic acidmolecules. These nucleic acid molecules hybridize to nucleic acidmolecules described in the preceding paragraphs.

The nucleic acid molecule of the invention may have a sense or anantisense sequence.

In another embodiment, the invention is an isolated oligonucleotideconsisting of a sequence selected from the group consisting of5′-GCCATGTTGGATTCTCTAGTGTCG-3 (SEQ ID NO:11),5′-CCGMTTCTCAAAGCTTTTCTTCCACG-3′ (SEQ ID NO:12),5′-CGGMTTCACAGAAAMCACAGTGAGGAT-3′ (SEQ ID NO:13), an oligonucleotidewith an antisense sequence of 5′-GCCATGTTGGATTCTCTAGTGTCG-3 (SEQ IDNO:14), an oligonucleotide with an antisense sequence of5′-CCGMTTCTCAAAGCTTTTCTTCCACG-3′ (SEQ ID NO:15) and an oligonucleotidewith an antisense sequence of 5′-CGGMTTCACAGAAAAACACAGTGAGGAT-3′ (SEQ IDNO:16). The invention includes an isolated oligonucleotide consisting of5 to 15 nucleotides of these oligonucleotides. The invention includes anisolated oligonucleotide consisting of a sequence homologous to theoligonucleotide of claim 15 or claim 16.

In an alternate embodiment, the invention is an expression vectorcomprising a nucleic acid molecule of the invention. The expressionvector preferably consists of a promoter selected from the groupconsisting of a super promoter, a 35S promoter of cauliflower mosaicvirus, a drought-inducible promoter, an ABA-inducible promoter, a heatshock-inducible promoter, a salt-inducible promoter, a copper-induciblepromoter, a steroid-inducible promoter and a tissue-specific promoter.

The invention is a polypeptide produced from the nucleic acid moleculesof the invention. The invention is also a polypeptide produced from theexpression vector. The polypeptide is used for extrusion of sodium ionsfrom the cytosol of a cell to provide the cell with salt tolerance.

In a preferred embodiment, the polypeptide has the amino acid sequencein FIG. 1( a)-(e). The polypeptides may be homologous to the polypeptidein FIG. 1( a)-(e). In an alternate embodiment, the polypeptides comprisea sequence having greater than 28% homology to the polypeptide in FIG.1( a)-(e). The polypeptides are Na⁺/H⁺ exchanger polypeptides.

The polypeptides are preferably isolated from Arabidopsis thaliana.

The invention includes peptides consisting of at least 5 amino acids ofthe polypeptides described in the preceding paragraphs. In anotherembodiment, the peptides consist of 41 to 75 amino acids of thepolypeptides described in the preceding paragraphs.

The invention also includes isolated nucleic acid molecules encoding thepolypeptides of the invention. The isolated nucleic acid moleculepreferably encodes the polypeptide of FIG. 1( a)-(e).

The polypeptides of the invention that extrude sodium ions from thecytosol of a cell to provide the cell with salt tolerance, preferablyconsist of an amiloride binding domain. The amiloride binding domain isbetween amino acids 82 to 90 in both AtNHX1 and AtNHX3 in FIG. 1( a)-(e)and between amino acids 58 to 66 in both AtNHX5 and AtNHX4 in FIGS. (d)and (e).

The invention also includes a monoclonal antibody or polyclonal antibodydirected against a polypeptide of the invention.

Another embodiment of the invention includes a transformed microorganismcomprising an isolated nucleic acid molecule of the invention. Theinvention also includes a transformed microorganism including anexpression vector.

The invention includes a plant cell transformed with a nucleic acidmolecule of the invention. The invention also includes a yeast celltransformed with the nucleic acid molecule of the invention. In anotherembodiment, the invention is a plant, plant part or seed, generated froma plant cell transformed with a nucleic acid molecule of the invention.The invention also relates to a plant, plant part, seed or plant celltransfected with a nucleic acid molecule of the invention. The plant,plant part, seed or plant cell is preferably selected from a speciesselected from the group consisting of potato, tomato, brassica, cotton,sunflower, strawberries, spinach, lettuce, rice, soybean, corn, wheat,rye, barley, atriplex, sorgum, alfalfa and salicornia and other plantsin Table 5.

The invention also includes a method for producing a polypeptide of theinvention by culturing a plant, plant part, seed or plant cell of theinvention and recovering the expressed polypeptide from the culture.

The invention includes an isolated nucleic acid molecule encoding apolypeptide capable of extruding monovalent cations from the cytosol ofa cell to provide the cell with increased salt tolerance. The nucleicacid molecule preferably includes the nucleotide sequence in FIG. 1(a)-(e). The nucleic acid molecule is preferably DNA or RNA. The cell ispreferably a plant cell, a yeast cell or a bacterial cell. Themonovalent cations are preferably sodium, lithium or potassium. Themonovalent cations are preferably extruded into a vacuole or out of thecell. The nucleic acid molecules preferably encode a Na⁺/H⁺ exchangerpolypeptide. The nucleic acid molecule is preferably isolated fromArabidopsis thaliana.

The invention also includes an isolated nucleic acid molecule, includinga sequence having greater than 17% homology to a sequence referred to inthe preceding paragraph.

The invention also includes a nucleic acid molecule of 8 to 10nucleotides, 11 to 25 or 26 to 50 nucleotides of a nucleic acid moleculeof the invention.

The invention also includes a nucleic acid molecule which nucleic acidmolecule hybridizes a nucleic acid molecule of the invention. Thenucleic acid molecule comprises a sense or an antisense sequence.

The invention also includes an isolated oligonucleotide including asequence selected from the group consisting of5′-GCCATGTTGGATTCTCTAGTGTCG-3′ (SEQ ID NO:11),5′-CCGAATTCTCAAAGCTTTTCTTCCACG-3′ (SEQ ID NO:12),5′-CGGAATTCACAGAAAAACACAGTGAGGAT-3′ (SEQ ID NO:13),5′-GCCATGTTGGATTCTCTAGTGTCG-3′ (SEQ ID NO:14),5′-CCGAATTCTCAAAGCTTTTCTTCCACG-3′ (SEQ ID NO:15),5′-CGGAATTCACAGAAAAACACAGTGAGGAT-3′ (SEQ ID NO:16),

or 5 to 15 nucleotides of one of these oligonucleotides. The inventionalso includes an isolated oligonucleotide having a sequence homologousto one of these oligonucleotides. The invention also includes anexpression vector including a nucleic acid molecule of the invention.The expression vector preferably comprises a promoter selected from thegroup consisting of a super promoter, a 35S promoter of cauliflowermosaic virus, a drought-inducible promoter, an ABA-inducible promoter, aheat shock-inducible promoter, a salt-inducible promoter, acopper-inducible promoter, a steroid-inducible promoter and atissue-specific promoter.

The invention also includes a polypeptide produced from a nucleic acidmolecule or expression vector of the invention. The invention alsoincludes a polypeptide for extrusion of monovalent cations ions from thecytosol of a cell to provide the cell with salt tolerance. The inventionalso includes a polypeptide including the amino acid sequence in FIG. 1(a)-(e) or a polypeptide homologous to one of these sequences. Theinvention also includes a polypeptide including a sequence havinggreater than 28% homology to one of these polypeptides. The polypeptideis preferably a Na⁺/H⁺ exchanger polypeptide isolated from Arabidopsisthaliana. The invention also includes a peptide including at least 5amino acids or 41 to 75 amino acids of the polypeptide of the invention.The invention also includes nucleic acid molecules these polypeptides.

The invention includes a polypeptide for extrusion of monovalent cationsions from the cytosol of a cell to provide the cell with salt tolerance,including, but not necessarily having, an amiloride binding domain.

Another aspect of the invention relates to a monoclonal or polyclonalantibody directed against a polypeptide of the invention.

Another variation includes a transformed microorganism including anisolated nucleic acid molecule of the invention. The transformedmicroorganism preferably includes an expression vector of the invention.

A plant cell, yeast cell transformed or transfected with a nucleic acidmolecule or a plant, plant part or seed, generated from the plant cell.The plant, plant part, seed or plant cell is preferably from a speciesselected from the group consisting of potato, tomato, brassica, cotton,sunflower, strawberries, spinach, lettuce, rice, soybean, corn, wheat,rye, barley, atriplex, sorgum, alfalfa, salicornia and other plants inTable 5. The invention also includes a method for producing a peptide,by culturing the plant, plant part, seed or plant cell and recoveringthe expressed peptide from the culture.

The invention includes a nucleic acid molecule that encodes all or partof a polypeptide capable of extruding monovalent cations from thecytosol of a cell to provide the cell with salt tolerance, wherein thesequence hybridizes to the nucleic acid molecule of all or part of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:19, FIG. 5( b) or anucleic acid molecule including nucleotides 1-1487 of FIG. 5( b) underlow, medium and high stringency conditions. The high stringencyconditions preferably comprise a wash stringency of selected from thegroup of hybridization and wash stringencies in Table 4.

The invention includes an isolated nucleic acid molecule encoding apolypeptide capable of extruding monovalent cations from the cytosol ofa cell to provide the cell with salt tolerance, including the nucleicacid molecule in FIG. 5( b). The invention also includes an isolatednucleic acid molecule encoding a polypeptide capable of extrudingmonovalent cations from the cytosol of a cell to provide the cell withsalt tolerance, including nucleotides 1 to 1487 of the nucleic acidmolecule in FIG. 5( b).

Another aspect of the invention relates to an isolated nucleic acidmolecule including about 1640 (or preferably about 1600 or 1700) nucleicacids encoding a polypeptide capable of extruding monovalent cationsfrom the cytosol of a cell to provide the cell with salt tolerance, thenucleic acid molecule including nucleotides 1 to 1487 (or preferablyabout nucleotides 1 to 1470, 1480, 1490 or 1500) of the nucleic acidmolecule in FIG. 5( b). The molecule is preferably DNA or RNA. The cellis preferably selected from the group consisting of a plant cell, ayeast cell and a bacterial cell. The molecule preferably encodes aNa⁺/H⁺ exchanger polypeptide. The nucleic acid molecule is preferablyisolated from Arabidopsis thaliana.

The invention also includes the nucleic acid molecule in FIG. 5( b) or anucleic acid molecule having greater than 17% homology to the sequencein 5(b). The invention includes polypeptides produced from this one ofthese nucleic acid molecules. The invention also relates to apolypeptide including the amino acid sequence in FIG. 5( b) or aminoacids 1 to 496 of FIG. 5( b). (note: polypeptide including 1 to 496 ispreferably about 530, 540 or 550 amino acids, most preferably about 538amino acids) or a homologous polypeptide, preferably having greater than28% homology. The polypeptide is preferably a Na⁺/H⁺ exchangerpolypeptide, isolated from Arabidopsis thaliana. The invention alsoincludes a DNA molecule encoding one of these polypeptides.

The invention includes an isolated nucleic acid molecule encoding apolypeptide capable of extruding monovalent cations from the cytosol ofa cell to provide the cell with salt tolerance, including at least oneof the nucleic acid molecules in FIG. 1( c). The molecule is preferablyDNA or RNA. The cell is preferably selected from the group consisting ofa plant cell, a yeast cell and a bacterial cell and encodes a Na⁺/H⁺exchanger polypeptide isolated from Arabidopsis thaliana.

The invention includes an isolated nucleic acid molecule, including thenucleic acid molecule in FIG. 1( c)(i) or 1(c)(ii) or a polypeptideproduced from a nucleic acid molecule of the invention. The inventionalso includes a polypeptide including the amino acid sequence in FIG. 1(c)(i) or 1(c)(ii) or homologous to this polypeptide, preferably havinggreater than 28% homology. The polypeptide is preferably a Na⁺/H⁺exchanger polypeptide, isolated from Arabidopsis thaliana. The inventionincludes a DNA molecule encoding one of these polypeptides.

It will be clear to one skilled in the art that the sequences in FIGS.1( c) and 5 are useful in isolating other salt tolerant nucleic acidmolecules (for example probes may be made from the sequences in FIGS. 1(c) and 5), preparing transgenic plants and performing many of the othermethods of the invention that are described with respect to sequences inFIGS. 1( a), (b), (d) and (e). Variants and modifications of FIG. 1( c)and FIG. 5 sequences are also included within the invention as aremethods using varied or modified sequences (the same preferredpercentages of identity and sequence described with respect to FIGS. 1(a), (b), (d) and (e) also apply to FIGS. 1( c) and 5). Nucleic acidmolecules including a portion of the nucleic acid molecule in FIG. 5preferably include about nucleotides 1-1487 (or a partial sequencethereof, preferably starting from the coding region, which will beapparent to a skilled person, at about nucleotide 286). The nucleotidesequence including all or part of sequence in FIG. 1( c) or FIG.(5) willbe preferably about 1614 nucleotides in length (or the 1614 nucleotidesminus part or all of the 5′ untranslated region nucleotides). Thenucleic acid molecules are most preferably 1600 to 1620 nucleotides inlength. Polypeptides including a portion of the nucleic acid molecule inFIG. 5 preferably include about amino acids 1 to 496 (or a partialsequence thereof) in FIG. 5. The sequences encoding all or part of thepolypeptide in FIG. 5 or encoding a polypeptide corresponding to eitherof the nucleic acid molecule sequences in FIG. 1( c) are preferablyabout 538 amino acids in length and preferably about 60 kda in length.Preferred polypeptides are about 530-550 amino acids in length.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Preferred embodiments of the invention are described in relation to thedrawings in which:

FIG. 1. (a) Shows the nucleic acid molecule that is SEQ ID NO:1 and thepolypeptide that is SEQ ID NO:2.

The figure shows isolated AtNHX1 cDNA encoding a Na+/H+ exchanger fromArabidopsis thaliana showing cDNA sequence and the corresponding aminoacid sequence for AtNHX1. Twelve transmembrane domains are present, aconserved amiloride-binding domain is present, and a relativelyhydrophilic C-terminal region is also present. The predicted openreading frame begins at nucleotide 286. The amino acids are centeredbelow the corresponding codon and are numbered on the left.

(b) Shows the nucleic acid molecule that is SEQ ID NO:3 and thepolypeptide that is SEQ ID NO:4.

The figure shows isolated AtNHX3 cDNA encoding a Na+/H+ exchanger fromArabidopsis thaliana showing cDNA sequence and the correspondingpredicted amino acid sequence for AtNHX3. The predicted open readingframe begins at nucleotide 61. The amino acids are centered below thecorresponding codon and are numbered on the left.

(c) (i) Shows the nucleic acid molecule that is SEQ ID NO:5 and thepolypeptide that is SEQ ID NO:6. (ii) Shows the nucleic acid moleculethat is SEQ ID NO:7 and the polypeptide that is SEQ ID NO:8.

The figure shows AtNHX5 partial cDNA sequences. The amino acids arecenetred below the corresponding codon and are numbered on the left (i)5′ sequence of the partial AtNHX5 cDNA and amino acid sequence; and (ii)the figure shows the 3′ sequence of the partial AtNHX5 cDNA and aminosequence.

(d) Shows the nucleic acid molecule that is SEQ ID NO:17 and thepolypeptide that is SEQ ID NO:18.

The figure shows isolated AtNHX5 cDNA encoding a Na+/H+ exchanger fromArabidopsis thaliana showing cDNA sequence and the correspondingpredicted amino acid sequence for AtNHX5. The predicted open readingframe begins at nucleotide 67. The amino acids are centered below thecorresponding codon and are numbered on the left. (e) Isolated AtNHX4cDNA encoding a Na+/H+ exchanger from Arabidopsis thaliana showing thecDNA sequence of SEQ ID NO:19 and the corresponding predicted amino acidsequence SEQ ID NO:20 for AtNHX4. The predicted open reading framebegins at nucleotide 55. The amino acids are centred below thecorresponding codon and are numbered on the left.

(e) Shows the nucleic acid molecule that is SEQ ID NO:19 and thepolypeptide that is SEQ ID NO:20.

The figure shows isolated AtNHX4 cDNA encoding a Na+/H+ exchanger fromArabidopsis thaliana.

FIG. 2. (a) Alignment of the predicted amino acid sequences ofArabidopsis AtNHX1 SEQ ID NO:2, from Arabidopsis thaliana with otherNa+/H+ exchangers from other organisims. Sequences were aligned usingthe Clustal W program (19) using default parameters (fixed gappenalty=10, floating gap penalty=10, protein weight matrix BLOSUM62).Sequences and GenBank accession numbers are: ScNHX1, late endosomalNa+/H+ exchanger S. cerevisiae, SEQ ID NO:29 (GenBank accession#927695); CeNHE1, C. elegans, SEQ ID NO:31 (GenBank accession #3877723;HsNHE6, Homo sapiens mitochondrial Na+/H+ exchanger, SEQ ID NO:30(GenBank accession #2944237); (b) Alignment of the predicted amino acidsequences of AtNHX1 SEQ ID NO:2, AtNHX3 SEQ ID NO:4 and AtNHX5 SEQ IDNO:18 cDNAs from Arabidopsis thaliana. Sequences were aligned using theClustal W program using default parameters (fixed gap penalty=10,floating gap penalty=10, protein weight matrix BLOSUM62); (c) Alignmentof the predicted amino acid sequences of AtNHX5 SEQ ID NO:18 and AtNHX4SEQ ID NO:20 cDNAs from Arabidopsis thaliana. Sequences were alignedusing the Clustal W program using default parameters (fixed gappenalty=10, floating gap penalty=10, protein weight matrix BLOSUM62).

FIG. 3. A Southern blot of Arabidopsis genomic DNA. Genomic DNA (10 μgper lane) was digested with various restriction enzymes, separated on a1.0% agarose gel, transferred onto a GeneScreen Plus membrane(Amersham), and hybridized to a radiolabelled AtNHX1 cDNA as describedin Materials and Methods. Restriction enzymes used were; C, ClaI; E,ECoRI; X, XbaI; H, HindIII.

FIG. 4. RNA blot of AtNHX1 expression in different tissues. Total RNA(40 μg) was separated on a 1.0% agarose gel, transferred to a GeneScreenPlus membrane (Amersham) and hybridized to a radiolabelled AtNHX1 cDNAprobe as described in Materials and Methods. Tissues in each lane wereas follows: 1, mature leaf; 2, flower (including sepals); 3,infloresence stem; 4, seedling shoot; 5, seedling root.

FIG. 5. (a) and (b) show the nucleic acid molecule that is SEQ ID NO:9and the polypeptide that is SEQ ID NO:10.

Figures (a) and (b) show modified Arabidopsis sodium/proton antiportercDNA and polypeptide sequence.

FIG. 6. RNA blot comparing transcript levels in Arabidopsis thalianaleaf tissue from wild type and different transgenic lines overexpressingAtNHX1. RNA was extracted from 4 week-old plants. Total RNA (30 μg perlane) was separated on a 1.0% agarose gel, transferred to a GeneScreenPlus membrane (Amersham) and hybridized to a radiolabelled AtNHX1 cDNAprobe as described in Materials and Methods. An endogenous 2.1 kbtranscript was detected in the transgenic lines as well as in wild type.An overexpressed 1.8 kb transcript was only seen in the transgeniclines. The 1.8 kb transcript corresponds to the open reading framecoding for AtNHX1, lacking the 5′- and 3′-untranslated regions presentin the original cDNA (2.1 kb). Ribosomal RNA (rRNA) was used to confirmequal loading of the gels, as seen by methylene-blue staining of theblot.

wt: wild-type; X1-2′, X1-3′ and X1-4′: independent transgenic lines.

FIG. 7. Twenty 3-week old kanamycin-resistant Arabidopsis thalianaplants for each of the 3 independent transgenic lines (X1.2′, X1.3′ andX1.4′) transformed with AtNHX1, as well as 20 wild-type plants of thesame age were used for assessment of salt tolerance. Plants were wateredwith 25 ml of ⅛ strength MS salts (control solution) supplemented withdifferent concentrations of NaCl. The following schedule was used for atotal of 16 days, at which point pictures of representative plants weretaken: a) wild-type: A=0 mM NaCl, B=50 mM NaCl, C=100 mM NaCl, D=150 mMNaCl, E=200 mM NaCl; b) X1.2′ transgenic line: A=0 mM NaCl, B=50 mMNaCl, C=100 mM NaCl, D=150 mM NaCl, E=200 mM NaCl; c) X1.3′ transgenicline; d) X1.4′ transgenic line: A=0 mM NaCl, B=50 mM NaCl, C=100 mMNaCl, D=150 mM NaCl, E=200 mM NaCl; e) wild type: A=0 mM NaCl, E=200 mMNaCl vs. transgenic strain 2′: A=0 mM NaCl, E=200 mM NaCl; f) wild type:A=0 mM NaCl, E=200 mM NaCl vs. transgenic strain 4′: A=0 mM NaCl, E=200mM NaCl; g) wild type: A=0 mM NaCl, E=200 mM NaCl vs. transgenic strain2′: A=0 mM NaCl, E=200 mM NaCl and transgenic strain 4′: A=0 mM NaCl,E=200 mM NaCl.

Treatments:

-   A) watered with a control solution (⅛ MS strength solution, 0 mM    NaCl) eight times (once every two days)-   B) watered with a control solution supplemented with 50 mM NaCl    eight times (once every two days)-   C) watered twice (once every two days) with a control solution    supplemented with 50 mM NaCl, then with a control solution    supplemented with 100 mM NaCl six times (once every two days).-   D) watered twice (once every two days) with a control solution    supplemented with 50 mM NaCl, then with a control solution    supplemented with 100 mM NaCl twice (once every two days) followed    by a control solution supplemented with 150 mM NaCl four times (once    every two days).-   E) watered twice (once every two days) with a control solution    supplemented with 50 mM NaCl, then with a control solution    supplemented with 100 mM NaCl twice (once every two days) followed    by a control solution supplemented with 150 mM NaCl twice (once    every two days) and a control solution supplemented with 200 mM NaCl    twice (once every two days).

FIG. 8. (a) shows SEQ ID NO:21 (b) shows SEQ ID NO:22 (c) shows SEQ IDNO:23 (d) shows SEQ ID NO:24 (e) shows SEQ ID NO:25 (f) shows SEQ IDNO:26 (g) shows SEQ ID NO:27 (h) shows SEQ ID NO:28.

Figures (a)-(h) show sequences from Table 2: (a) GenBank Accession No.3850064 569 a.a.; (b) GenBank Accession No. 927695 633 a.a.; (c) GenBankAccession No. C91832 378 bp mRNA EST; (d) GenBank Accession No. C91861268 bp mRNA EST; (e) GenBank Accession No. AU032544 380 bp mRNA EST; (f)GenBank Accession No. AA660573 596 bp mRNA EST; (g) GenBank AccessionNo. L44032 522 bp mRNA STS; (h) GenBank Accession No. T75860 (EST) 330bp mRNA EST.

DETAILED DESCRIPTION OF THE INVENTION

Salt Tolerance Nucleic Acid Molecules and Polypeptides

The invention relates to nucleic acid molecules and polypeptides whichincrease salt tolerance in cells and plants. PNHX polypeptides are plantNa⁺/H⁺ transporter polypeptides that are capable of increasing andenhancing salt tolerance in a cell, preferably a plant cell. Thesetransporters (also referred to as exchangers, antiports or antiporters)extrude monovalent cations (preferably potassium ions or lithium ions,most preferably sodium ions) out of the cytosol. The cations arepreferably extruded into the vacuoles or extracellular space. Theaffinity for particular ions varies between transporters. The listedpreferences refer to the cations that are most likely to be abundant inthe cytosol and therefore most likely to be extruded. It is notnecessarily a reflection of transporter affinity for particular cations.The PNHX nucleic acid molecules which encode PNHX polypeptides areparticularly useful in producing transgenic plants which have increasedsalt tolerance compared to a wild type plant.

It will also be apparent that there are polypeptide and nucleic acidmolecules from other organisms, such as yeast, microorganisms, fish,birds or mammals, that are similar to PNHX polypeptides and nucleic acidmolecules. The entire group of Na⁺/H⁺ transporter polypeptides andnucleic acid molecules that are capable of increasing salt tolerance ina cell (including PNHX and AtNHX polypeptides and nucleic acidmolecules) are collectively referred to as (“TNHX polypeptides” and“TNHX nucleic acid molecules”). TNHX polypeptides are Na⁺/H⁺transporters that are capable of increasing salt tolerance in a cell,preferably a plant cell, because they extrude monovalent cations(preferably potassium ions or lithium ions, most preferably sodium ions)out of the cytosol.

The role of TNHX and PNHX nucleic acid molecules and polypeptides inmaintaining salt tolerance was not shown before this invention. Theability of these compounds to increase salt tolerance of transgenic hostcells (particularly plant cells) and transgenic plants compared to wildtype cells and plants was unknown.

PNHX and TNHX polypeptides need not necessarily have the primaryfunction of providing salt tolerance. All nucleotides and polypeptideswhich are suitable for use in the methods of the invention, such as thepreparation of transgenic host cells or transgenic plants, are includedwithin the scope of the invention. Genomic clones or cDNA clones arepreferred for preparation of transgenic cells and plants.

In a preferred embodiment, the invention relates to cDNAs encodingNa+/H+ exchangers from Arabidopsis thaliana. The cDNA sequences and thecorresponding amino acid sequences for AtNHX1, AtNHX3, AtNHX4 and AtNHX5are presented in FIG. 1. AtNHX1 and AtNHX3 are homologs that arephysically located at different places in the genome. The invention alsoincludes splice variants of the nucleic acid molecules as well aspolypeptides produced from the molecules. For example, AtNHX5 and AtNHX4are homologs of AtNHX1 and AtNHX3. AtNHX5 and AtNHX4 are identical for along sequence beginning at the N-terminus. This indicates that thedifference in sequence at the C-terminus is due to alternative splicingof a nucleic acid molecule (also known as splicing variants). Thisallows a single nucleic acid molecule to produce varying polypeptides.

Characterization of Salt Tolerance Nucleic Acid Molecules andPolypeptides

The longest open reading frame of 1614 base pairs in AtNHX1 encodes apolypeptide of 538 amino acids with a predicted molecular weight (“MW”)of about 60 Kda. A comparison of this full length cDNA with theArabidopsis genome sequence (A-TM021B04.4) revealed the presence of 13introns and 14 exons. This polypeptide encoded by the open reading framewas about 19% larger than the sequence predicted by the Arabidopsisgenomic sequence (A-TM021B04.4). This sequence encodes the full lengthexchanger given that the cDNA region immediately upstream of the startcodon contains predicted stop codons in all three reading frames. Inaddition, a transcript of approximately 2 kb, which corresponds roughlyin size to the predicted mRNA for AtNHX1, was observed on RNA blots.Based on the amino acid sequence of AtNHX1, 12 transmembrane domains arepredicted, a conserved amiloride-binding domain is present, and arelatively hydrophilic C-terminal region is also predicted. AtNHX1 showssome similarity at the amino acid level to Na+/H+ exchangers isolatedfrom a variety of organisms ranging from yeast (about 27% identity) tohumans (about 20%). A second salt tolerance cDNA and polypeptide,AtNHX3, was obtained from Arabidopsis thaliana (FIG. 1( b)). Wecharacterized a third salt tolerance nucleic acid molecule, AtNHX5, byobtaining 5′ and 3′ cDNA and N-terminal and C-terminal sequences fromArabidopsis thaliana (FIG. 1( c)). In one variation, the inventionincludes DNA sequences (and the corresponding polypeptide) including atleast one of the sequences shown in FIG. 1( c) in a nucleic acidmolecule of preferably about: less than 1000 base pairs, less than 1250base pairs, less than 1500 base pairs, less than 1750 base pairs, lessthan 2000 base pairs, less than 2250 base pairs, less than 2500 basepairs, less than 2750 base pairs or less than 3000 base pairs. We alsoidentified the full AtNHX5 sequence (FIG. 1( d)). A fourth sequence,AtNHX4, was also identified (FIG. 1( e)).

The coding regions of the nucleic acid molecules are as follows:

TABLE 1 Nucleic Acid Molecule Start Nucleotide End Nucleotide AtNHX1 2861902 AtNHX3 61 1707 AtNHX5 67 1024 AtNHX4 55 813

It will be apparent that these may be varied, for example, by shorteningthe 5′ untranslated region or shortening the nucleic acid molecule sothat the end nucleotide is in a different position.

The discussion of the nucleic acid molecules, sequence identity,hybridization and other aspects of nucleic acid molecules includedwithin the scope of the invention is intended to be applicable to eitherthe entire nucleic acid molecules in FIGS. 1( a), (b), (d) and (e) andthe coding regions of these molecules, shown in Table 1. One may use theentire molecule in FIG. 1 or only the coding region. Other possiblemodifications to the sequence will also be apparent.

Southern Blot Analysis (FIG. 3) suggests that AtNHX1 is likely presentas a single copy gene in Arabidopsis. A Northern blot (FIG. 4) showedthat AtNHX polypeptide (particularly AtNHX1) was expressed in alltissues examined (root, shoot (shoot includes leaves and stems), flower,inflorescence stem).

Function of Salt Tolerance Nucleic Acid Molecules

The polypeptides of the invention allow the extrusion of monovalentcations (preferably potassium ions or lithium ions, most preferablysodium ions) from the cytosol, which in this application preferablyrefers to the transport and accumulation of sodium ions into thevacuoles or into the extracellular space (outside of the cell), thusproviding the most important trait for salt tolerance in plants.Antiport polypeptides from organisms other than plants have showndifferent specificity for monovalent ions (e.g. D. G. Warnock, A. S.Pollock, “Sodium Proton Exchange in Epithelial Cells”, pages 77-90, inS. Grinstein ed. Sodium Proton Exchange, (1987, CRC Press, USA).) TNHXand PNHX transporters will also show different specificity betweentransporters. The nucleic acid molecules of the invention allow theengineering of salt tolerant plants by transformation of crops with thisnucleic acid molecule under the control of constitutively activepromoters or under the control of conditionally-inducible promoters. Theresulting expression or overexpression of these nucleic acid moleculesconfers increased salt tolerance in plants grown in soil, solid,semi-solid medium or hydroponically.

The PNHX Nucleic Acid Molecule and Polypeptide is Conserved in Plants

Sequence Identity

This is the first isolation of a nucleic acid molecule encoding a Na⁺/H⁺exchanger from plant species. It is widely known amongst those skilledin the art that Arabidopsis thaliana is a model plant for many plantspecies. Nucleic acid sequences having sequence identity to the AtNHXsequences are found in other plants, in particular halophytes such asBeta Vulgaris and Atriplex (see Examples 2 and 7). Sequences fromArabidopsis thaliana and other plants are collectively referred to as“PNHX” nucleic acid sequences and polypeptides. We isolate PNHX nucleicacid molecules from plants having nucleic acid molecules that aresimilar to those in Arabidopsis thaliana, such as beet, tomato, rice,cucumber, radish and other plants as in Table 5 and using techniquesdescribed in this application. The invention includes methods ofisolating these nucleic acid molecules and polypeptides as well asmethods of using these nucleic acid molecules and polypeptides accordingto the methods described in this application, for example those usedwith respect to AtNHX.

Table 2 below shows several sequences with sequence identity andsequence similarity to the AtNHX polypeptides. Where polypeptides areshown, a suitable corresponding DNA encoding the polypeptide will beapparent. These sequences code for polypeptides similar to portions ofAtNHX polypeptides. The sequences in Table 2 are useful to make probesto identity full length sequences or fragments (from the listed speciesor other species). One skilled in the art would be able to design aprobe based on a polypeptide or peptide fragment. The invention includesnucleic acid molecules of about: 10 to 50 nucleotides, 50 to 200nucleotides, 200 to 500 nucleotides, 500 to 1000 nucleotides, 1000 to1500 nucleotides, 1500 to 1700 nucleotides, 1700 to 2000 nucleotides,2000 to 2500 nucleotides or at least 2500 nucleotides and which includeall or part of the sequences (or corresponding nucleic acid molecule) inTable 2. The invention also includes peptides and polypeptides of about:10 to 50 amino acids, 50 to 200 amino acids, 200 to 500 amino acids, 500to 750 amino acids or at least 750 amino acids which encode all or partof the polypeptides in Table 2 (wherein the polypeptide is producedaccording to a reading frame aligned with an AtNHX polypeptide).Possible modifications to these sequences will also be apparent. Thepolypeptide and nucleic acid molecules are also useful in researchexperiments or in bioinformatics to locate other sequences. The nucleicacid molecules and polypeptides preferably provide Na⁺/H⁺ transporteractivity and are capable of moving monovalent cations from the cytosolof the cell into vacuoles or the extracellular space (in thisapplication, extracellular space refers to the space outside a cell inan organism or the space outside a cultured cell).

TABLE 2 Organism GenBank Accession No. Yeast (S. pombe) (FIG. 8(a))3850064 Yeast (Saccharomyces cervisae) (FIG. 8(b))  927695 Rice EST(FIG. 8(c)) C 91832 Rice EST (FIG. 8(d)) C 91861 Rice EST (FIG. 8(e))AV032544 Medicago Trunculata EST (FIG. 8(f)) AA660573 Hordeum VulgareSTS (FIG. 8g)) L 44032

As shown in Table 3 below, many nucleic acid molecules identified inArabidopsis thaliana have striking DNA sequence similarity to nucleicacid molecules encoding the homologous polypeptide in other plantspecies. Using the techniques described in this application and othersknown in the art, it will be apparent that the nucleic acid moleculeencoding the homologous Na⁺/H⁺ exchanger in other plant speciesincluding, but not limited to plants of agricultural and commercialinterest, will have DNA sequence identity (homology) at leastabout >17%, >20%, >25%, >35% to a DNA sequence shown in FIG. 1 or 5 (ora partial sequence thereof). Some plants species may have DNA with asequence identity (homology) at least about: >50%, >60%, >70%, >80%or >90% more preferably at least about >95%, >99% or >99.5%, to a DNAsequence in FIG. 1 or 5 (or a partial sequence thereof). The inventionalso includes modified nucleic acid molecules from plants other thanArabidopsis thaliana which have sequence identity at leastabout: >17%, >20%, >25%, >35%, >50%, >60%, >70%, >80% or >90% morepreferably at least about >95%, >99% or >99.5%, to an AtNHX sequence inFIG. 1 or 5 (or a partial sequence thereof). Modified nucleic acidmolecules are discussed below. Preferably about 1, 2, 3, 4, 5, 6 to 10,10 to 25, 26 to 50 or 51 to 100, or 101 to 250 nucleotides or aminoacids are modified. Sequence identity is most preferably calculated asthe number of identical amino acid residues expressed as a percentage ofthe length of the shorter of the two sequences in a pairwise alignment.The pairwise alignment is constructed preferably using the Clustal Wprogram preferably using the following parameter settings: fixed gappenalty=10, floating gap penalty=10, protein weight matrix=BLOSUM62. Forexample, if a nucleotide sequence (called “Sequence A”) has 90% identityto a portion of the nucleotide sequence in FIG. 1( a), then Sequence Awill be identical to the referenced portion of the nucleotide sequencein FIG. 8, except that Sequence A may include up to 10 point mutations,such as substitutions with other nucleotides, per each 100 nucleotidesof the referenced portion of the nucleotide sequence in FIG. 8.Polypeptides having sequence identity may be similarly identified.

The invention also includes nucleic acid molecules encoding polypeptideshaving sequence similarity taking into account conservative amino acidsubstitutions. Sequence similarity (and preferred percentages) arediscussed below.

It will be apparent that nucleic acid molecule encoding the homologousNa⁺/H⁺ exchanger in other species (preferably plants) including, but notlimited to plants of agricultural and commercial interest, willhybridize to all or part of a sequence in FIG. 1 or 5 (or a partialsequence thereof) under low, moderate (also called intermediateconditions) or high stringency conditions. Preferred hybridizationconditions are described below.

The invention includes the nucleic acid molecules from other plants aswell as methods of obtaining the nucleic acid molecules by, for example,screening a cDNA library or other DNA collection with a probe of theinvention (such as a probe comprising at least about: 10 or preferablyat least 15 or 30 nucleotides of AtNHX1, AtNHX3, AtNHX5 or AtNHX4 or asequence in FIG. 5) and detecting the presence of a TNHX or PNHX nucleicacid molecule. Another method involves comparing the AtNHX sequences (egin FIG. 1 or 5) to other sequences, for example using bioinformaticstechniques such as database searches or alignment strategies, anddetecting the presence of a TNHX or PNHX nucleic acid molecule orpolypeptide. The invention includes the nucleic acid molecule and/orpolypeptide obtained according to the methods of the invention. Theinvention also includes methods of using the nucleic acid molecules, forexample to make probes, in research experiments or to transform hostcells or make transgenic plants. These methods are as described below.

The polypeptides encoded by the homologous TNHX or PNHX nucleic acidmolecules in other species will have amino acid sequence identity. Thepreferred percentage of sequence identity for sequences of the inventionincludes sequences having identity of at least about: 30% to AtNHX1, 31%to AtNHX3, 36% to AtNHX5, and 36% to AtNHX4. Sequence identity may be atleast about: >20%, >25%, >28%, >30%, >35%, >40%, >50% to an amino acidshown in FIG. 1 or 5 (or a partial sequence thereof). Some polypeptidesmay have a sequence identity of at least about: >60%, >70%, >80%or >90%, more preferably at least about: >95%, >99% or >99.5% to anamino acid sequence in FIG. 1 or 5 (or a partial sequence thereof).Identity is calculated according to methods known in the art. Sequenceidentity is most preferably assessed by the Clustal W program. Theinvention also includes modified polypeptides from plants which havesequence identity at leastabout: >20%, >25%, >28%, >30%, >35%, >40%, >50%, >60%, >70%, >80%or >90% more preferably at least about >95%, >99% or >99.5%, to an AtNHXsequence in FIG. 1 or 5 (or a partial sequence thereof). Modifiedpolypeptides molecules are discussed below. Preferably about: 1, 2, 3,4, 5, 6 to 10, 10 to 25, 26 to 50 or 51 to 100, or 101 to 250nucleotides or amino acids are modified.

TABLE 3 Polypeptide DNA Plant Vacuolar H⁺-PPiase (vacuolarpyrophosphatase) Arabidopsis (Accession # 282878)  100%  100% Beet(Accession # 485742) 88.7% 72.8% Tobacco (Accession # 1076627) 89.9%68.4% Rice (Accession # 1747296)   85% 70.4% Tonoplast IntrinsicPolypeptide (water channel) Arabidopsis (Accession # X63551)  100%  100%Curcubita (Cucumber) (Accession # D45078) 66.5% 39.1% Raphanus (radish)(Accession # D84669) 56.7% 37.4% Helianthus (Accession # X95951) 50.4%35.2% High Affinity Ammonium Transporter Arabidopsis (Accession #X75879)  100%  100% Tomato (Accession # X95098) 73.5% 62.9% Rice(Accession # AF001505) 66.6% 58.1%Nucleic Acid Molecules and Polypeptides Similar to AtNHX

Those skilled in the art will recognize that the nucleic acid moleculesequences in FIGS. 1( a), (b), (d) and (e) are not the only sequenceswhich may be used to provide increased salt tolerance in plants. Thegenetic code is degenerate so other nucleic acid molecules which encodea polypeptide identical to an amino acid sequence in FIG. 1( a), (b),(d) or (e) may also be used. The sequence of the other nucleic acidmolecules of this invention may also be varied without changing thepolypeptide encoded by the sequence. Consequently, the nucleic acidmolecule constructs described below and in the accompanying examples forthe preferred nucleic acid molecules, vectors, and transformants of theinvention are merely illustrative and are not intended to limit thescope of the invention.

The sequences of the invention can be prepared according to numeroustechniques. The invention is not limited to any particular preparationmeans. For example, the nucleic acid molecules of the invention can beproduced by cDNA cloning, genomic cloning, DNA synthesis, polymerasechain reaction (PCR) technology, or a combination of these approaches((31) or Current Protocols in Molecular Biology (F. M. Ausbel et al.,1989).). Sequences may be synthesized using well known methods andequipment, such as automated synthesizers. Nucleic acid molecules may beamplified by the polymerase chain reaction. Polypeptides may, forexample, be synthesized or produced recombinantly.

Sequence Identity

The invention includes modified nucleic acid molecules with a sequenceidentity at least about: >17%, >20%, >30%, >40%, >50%, >60%, >70%, >80%or >90% more preferably at least about >95%, >99% or >99.5%, to a DNAsequence in FIG. 1 or 5 (or a partial sequence thereof). Preferablyabout 1, 2, 3, 4, 5, 6 to 10, 10 to 25, 26 to 50 or 51 to 100, or 101 to250 nucleotides or amino acids are modified. Identity is calculatedaccording to methods known in the art. Sequence identity is mostpreferably assessed by the Clustal W program. For example, if anucleotide sequence (called “Sequence A”) has 90% identity to a portionof the nucleotide sequence in FIG. 1( a), then Sequence A will beidentical to the referenced portion of the nucleotide sequence in FIG.1, except that Sequence A may include up to 10 point mutations, such asdeletions or substitutions with other nucleotides, per each 100nucleotide of the referenced portion of the nucleotide sequence inFIG. 1. Nucleotide sequences functionally equivalent to the PNHX orAtNHX sequences can occur in a variety of forms as described below.Polypeptides having sequence identity may be similarly identified.

The polypeptides encoded by the homologous NHX, PHX Na⁺/H⁺ exchangenucleic acid molecule in other species will have amino acid sequenceidentity (also known as homology) at leastabout: >20%, >25%, >28%, >30%, >40% or >50% to an amino acid sequenceshown in FIG. 1 or 5 (or a partial sequence thereof). Some plantsspecies may have polypeptides with a sequence identity (homology) of atleast about: >60%, >70%, >80% or >90%, more preferably at leastabout: >95%, >99% or >99.5% to all or part of an amino acid sequence inFIG. 1 or 5 (or a partial sequence thereof). Identity is calculatedaccording to methods known in the art. Sequence identity is mostpreferably assessed by the Clustal W program. Preferably about: 1, 2, 3,4, 5, 6 to 10, 10 to 25, 26 to 50 or 51 to 100, or 101 to 250nucleotides or amino acids are modified.

The invention includes nucleic acid molecules with mutations that causean amino acid change in a portion of the polypeptide not involved inproviding salt tolerance and ion transport or an amino acid change in aportion of the polypeptide involved in providing salt tolerance so thatthe mutation increases or decreases the activity of the polypeptide.

Hybridization

Other functional equivalent forms of the AtNHX nucleic acid moleculesencoding nucleic acids can be isolated using conventional DNA-DNA orDNA-RNA hybridization techniques. These nucleic acid molecules and theAtNHX sequences can be modified without significantly affecting theiractivity.

The present invention also includes nucleic acid molecules thathybridize to one or more of the sequences in FIG. 1 or 5 (or a partialsequence thereof) or their complementary sequences, and that encodeexpression for peptides or polypeptides exhibiting substantiallyequivalent activity as that of an AtNHX polypeptide produced by the DNAin FIG. 1 or their variants. Such nucleic acid molecules preferablyhybridize to the sequences under low, moderate (intermediate), or highstringency conditions. (see Sambrook et al. (Most recent edition)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). Preferable hybridization conditionsare about those in Table 4.

The present invention also includes nucleic acid molecules from anysource, whether modified or not, that hybridize to genomic DNA, cDNA, orsynthetic DNA molecules that encode the amino acid sequence of an AtNHXpolypeptide, or genetically degenerate forms, under salt and temperatureconditions equivalent to those described in this application, and thatcode for a peptide, polypeptide or polypeptide that has Na⁺/H⁺transporter activity. Preferably the polypeptide has the same or similaractivity as that of an AtNHX polypeptide. The nucleic acid molecules mayencode TNHX or PNHX polypeptides. A nucleic acid molecule describedabove is considered to be functionally equivalent to an AtNHX nucleicacid molecule (and thereby having Na⁺/H⁺ transporter activity) of thepresent invention if the polypeptide produced by the nucleic acidmolecule displays the following characteristics: the polypeptidemediates the proton-dependent sodium transport and sodium-dependentproton transport in intact cells, isolated organelles and purifiedmembrane vesicles. These sodium/proton movements should be higher(preferably at least about 50% higher and most preferably at least about100% higher) than the proton movements observed in the presence of abackground of potassium ions and/or other monovalent cations (i.e.rubidium, cesium, etc., but most preferably not lithium) (13,14).

The invention also includes nucleic acid molecules and polypeptideshaving sequence similarity taking into account conservative amino acidsubstitutions. Sequence similarity (and preferred percentages) arediscussed below.

Modifications to Nucleic Acid Molecule or Polypeptide Sequence

Changes in the nucleotide sequence which result in production of achemically equivalent or chemically similar amino acid sequences areincluded within the scope of the invention. Variants of the polypeptidesof the invention may occur naturally, for example, by mutation, or maybe made, for example, with polypeptide engineering techniques such assite directed mutagenesis, which are well known in the art forsubstitution of amino acids. For example, a hydrophobic residue, such asglycine can be substituted for another hydrophobic residue such asalanine. An alanine residue may be substituted with a more hydrophobicresidue such as leucine, valine or isoleucine. A negatively chargedamino acid such as aspartic acid may be substituted for glutamic acid. Apositively charged amino acid such as lysine may be substituted foranother positively charged amino acid such as arginine.

Therefore, the invention includes polypeptides having conservativechanges or substitutions in amino acid sequences. Conservativesubstitutions insert one or more amino acids which have similar chemicalproperties as the replaced amino acids. The invention includes sequenceswhere conservative substitutions are made that do not destroy Na+/H+transporter activity of the transporter polypeptide. The preferredpercentage of sequence similarity for sequences of the inventionincludes sequences having at least about: 48% similarity to AtNHX1, 48%similarity to AtNHX3, 56% similarity to AtNHX5, and 56% similarity toAtNHX4. The similarity may also be at least about: 60% similarity, 75%similarity, 80% similarity, 90% similarity, 95% similarity, 97%similarity, 98% similarity, 99% similarity, or more preferably at leastabout 99.5% similarity, wherein the polypeptide Na+/H+ has transporteractivity. The invention also includes nucleic acid molecules encodingpolypeptides, with the polypeptides having at least about: at leastabout: 48% similarity to AtNHX1, 48% similarity to AtNHX3, 56%similarity to AtNHX5, and 56% similarity to AtNHX4. The similarity mayalso be at least about: 60% similarity, 75similarity, 80% similarity,90% similarity, 95% similarity, 97% similarity, 98% similarity, or morepreferably at least about 99.5% similarity, wherein the polypeptideNa⁺/H⁺ has transporter activity, to an amino acid sequence in FIG. 1 or5 (or a partial sequence thereof) considering conservative amino acidchanges, wherein the polypeptide has Na+/H+ transporter activity.Sequence similarity is preferably calculated as the number of similaramino acids in a pairwise alignment expressed as a percentage of theshorter of the two sequences in the alignment. The pairwise alignment ispreferably constructed using the Clustal W program, using the followingparameter settings: fixed gap penalty=10, floating gap penalty=10,protein weight matrix=BLOSUM62. Similar amino acids in a pairwisealignment are those pairs of amino acids which have positive alignmentscores defined in the preferred protein weight matrix (BLOSUM62). Theprotein weight matrix BLOSUM62 is considered appropriate for thecomparisons described here by those skilled in the art ofbioinformatics. (The reference for the clustal w program (algorithm) isThompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W:improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, positions-specific gap penalties and weightmatrix choice. Nucleic Acids Research, 22:4673-4680; and the referencefor BLOSUM62 scoring matrix is Henikoff, S. and Henikoff, J. G. (1993)Performance evaluation of amino acid substitution matrices. Proteins,7:49-61.)

Polypeptides comprising one or more d-amino acids are contemplatedwithin the invention. Also contemplated are polypeptides where one ormore amino acids are acetylated at the N-terminus. Those of skill in theart recognize that a variety of techniques are available forconstructing polypeptide mimetics with the same or similar desiredbiological activity (Na⁺/H⁺ transporter activity) as the correspondingpolypeptide compound of the invention but with more favorable activitythan the polypeptide with respect to solubility, stability, and/orsusceptibility to hydrolysis and proteolysis. See, for example, Morganand Gainor, Ann. Rep. Med. Chem., 24:243-252 (1989). Examples ofpolypeptide mimetics are described in U.S. Pat. No. 5,643,873. Otherpatents describing how to make and use mimetics include, for example in,U.S. Pat. Nos. 5,786,322, 5,767,075, 5,763,571, 5,753,226, 5,683,983,5,677,280, 5,672,584, 5,668,110, 5,654,276, 5,643,873. Mimetics of thepolypeptides of the invention may also be made according to othertechniques known in the art. For example, by treating a polypeptide ofthe invention with an agent that chemically alters a side group byconverting a hydrogen group to another group such as a hydroxy or aminogroup. Mimetics preferably include sequences that are either entirelymade of amino acids or sequences that are hybrids including amino acidsand modified amino acids or other organic molecules.

The invention also includes hybrid nucleic acid molecules andpolypeptides, for example where a nucleotide sequence from one speciesof plant is combined with a nucleotide sequence from another sequence ofplant, mammal or yeast to produce a fusion polypeptide. The inventionincludes a fusion protein having at least two components, wherein afirst component of the fusion protein comprises a polypeptide of theinvention, preferably a full length AtNHX polypeptide. The secondcomponent of the fusion protein preferably comprises a tag, for exampleGST, an epitope tag or an enzyme. The fusion protein may comprise lacZ.

The invention also includes polypeptide fragments of the polypeptides ofthe invention which may be used to confer salt tolerance if thefragments retain Na⁺/H⁺ transporter activity. The invention alsoincludes polypeptides fragments of the polypeptides of the inventionwhich may be used as a research tool to characterize the polypeptide orits activity. Such polypeptides preferably consist of at least 5 aminoacids. In preferred embodiments, they may consist of 6 to 10, 11 to 15,16 to 25, 26 to 50, 51 to 75, 76 to 100 or 101 to 250 amino acids of thepolypeptides of the invention (or longer amino acid sequences). Thefragments preferably have sodium/proton transporter activity. Fragmentsmay include sequences with one or more amino acids removed, for example,C-terminus amino acids in an AtNHX sequence.

The invention also includes a composition comprising all or part of anisolated TNHX or PNHX (preferably AtNHX) nucleic acid molecule of theinvention and a carrier, preferably in a composition for planttransformation. The invention also includes a composition comprising anisolated TNHX or PNHX polypeptide (preferably AtNHX) and a carrier,preferably for studying polypeptide activity.

Recombinant Nucleic Acid Molecules

The invention also includes recombinant nucleic acid moleculescomprising a nucleic acid molecule of the invention and a promotersequence, operatively linked so that the promoter enhances transcriptionof the nucleic acid molecule in a host cell (the nucleic acid moleculesof the invention may be used in an isolated native gene or a chimericgene (for example, where a nucleic acid molecule coding region isconnected to one or more heterologous sequences to form a gene). Thepromoter sequence is preferably a constitutive promoter sequence or aninducible promoter sequence, operatively linked so that the promoterenhances transcription of the DNA molecule in a host cell. The promotermay be of a type not naturally associated with the cell. Transcriptionis enhanced with promoters known in the art such as the “Super-promoter”(20) or the ³⁵S promoter of cauliflower mosaic virus (21).

Inducible promoters are also used. These include:

-   -   a) drought- and ABA-inducible promoters which may include        ABA-responsive elements (22,23)    -   b) heat shock-inducible promoters which may contain HSEs (heat        shock elements) as well as CCAAT box sequences (24)    -   c) salt-inducible promoters which may include AT and PR elements        (25)    -   d) Copper-inducible promoter that includes ACE1 binding sites        (26)    -   e) steroid-inducible promoter that includes the glucocorticoid        response element along with an expression vector coding for a        mammalian steroid receptor (27).

In addition, tissue specific expression is achieved with the use oftissue-specific promoters such as, the Fd (Ferredoxin) promoter thatmediates high levels of expression in green leaves (28) and peroxidasepromoter for root-specific expression (29). These promoters vary intheir transcription initiation rate and/or efficiency.

A recombinant nucleic acid molecule for conferring salt tolerance mayalso contain suitable transcriptional or translational regulatoryelements. Suitable regulatory elements may be derived from a variety ofsources, and they may be readily selected by one with ordinary skill inthe art. Examples of regulatory elements include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the vector employed, other genetic elements,such as selectable markers, may be incorporated into the recombinantmolecule. Markers facilitate the selection of a transformed host cell.Such markers include genes associated with temperature sensitivity, drugresistance, or enzymes associated with phenotypic characteristics of thehost organisms.

Nucleic acid molecule expression levels are controlled with atranscription initiation region that regulates transcription of thenucleic acid molecule or nucleic acid molecule fragment of interest in aplant, bacterial or yeast cell. The transcription initiation region maybe part of the construct or the expression vector. The transcriptioninitiation domain or promoter includes an RNA polymerase binding siteand an mRNA initiation site. Other regulatory regions that may be usedinclude an enhancer domain and a termination region. The regulatoryelements described above may be from animal, plant, yeast, bacterial,fungal, viral or other sources, including synthetically producedelements and mutated elements.

Methods of modifying DNA and polypeptides, preparing recombinant nucleicacid molecules and vectors, transformation of cells, expression ofpolypeptides are known in the art. For guidance, one may consult thefollowing U.S. Pat. Nos. 5,840,537, 5,850,025, 5,858,719, 5,710,018,5,792,851, 5,851,788, 5,759,788, 5,840,530, 5,789,202, 5,871,983,5,821,096, 5,876,991, 5,422,108, 5,612,191, 5,804,693, 5,847,258,5,880,328, 5,767,369, 5,756,684, 5,750,652, 5,824,864, 5,763,211,5,767,375, or 5,750,848. Many of these patents also provide guidancewith respect to experimental assays, probes and antibodies,transformation of host cells and regeneration of plants, which aredescribed below. These patents, like all other patents, publications(such as articles and Genbank publications) in this application, areincorporated by reference in their entirety.

Host Cells Including a Salt Tolerance Nucleic Acid Molecule

In a preferred embodiment of the invention, a plant or yeast cell istransformed with a nucleic acid molecule of the invention or a fragmentof a nucleic acid molecule and inserted in a vector.

Another embodiment of the invention relates to a method of transforminga host cell with a nucleic acid molecule of the invention or a fragmentof a nucleic acid molecule, inserted in a vector. The invention alsoincludes a vector comprising a nucleic acid molecule of the invention.The TNHX, PNHX and AtNHX nucleic acid molecules can be cloned into avariety of vectors by means that are well known in the art. Therecombinant nucleic acid molecule may be inserted at a site in thevector created by restriction enzymes. A number of suitable vectors maybe used, including cosmids, plasmids, bacteriophage, baculoviruses andviruses. Suitable vectors are capable of reproducing themselves andtransforming a host cell. The invention also relates to a method ofexpressing polypeptides in the host cells. A nucleic acid molecule ofthe invention may be used to transform virtually any type of plant,including both monocots and dicots. The expression host may be any cellcapable of expressing TNHX, PNHX, such as a cell selected from the groupconsisting of a seed (where appropriate), plant cell, bacterium, yeast,fungus, protozoa, algae, animal and animal cell.

Levels of nucleic acid molecule expression may be controlled withnucleic acid molecules or nucleic acid molecule fragments that code foranti-sense RNA inserted in the vectors described above.

Agrobacterium tumefaciens-mediated transformation,particle-bombardment-mediated transformation, direct uptake,microinjection, coprecipitation and electroporation-mediated nucleicacid molecule transfer are useful to transfer a Na⁺/H⁺ transporternucleic acid molecule into seeds (where appropriate) or host cells,preferably plant cells, depending upon the plant species. The inventionalso includes a method for constructing a host cell capable ofexpressing a nucleic acid molecule of the invention, the methodcomprising introducing into said host cell a vector of the invention.The genome of the host cell may or may not also include a functionalTNHX or PNHX gene. The invention also includes a method for expressing aTNHX or PNHX transporter polypeptide in the host cell or a plant, plantpart, seed or plant cell of the invention, the method comprisingculturing the host cell under conditions suitable for gene expression.The method preferably also includes recovering the expressed polypeptidefrom the culture.

The invention includes the host cell comprising the recombinant nucleicacid molecule and vector as well as progeny of the cell. Preferred hostcells are fungal cells, yeast cells, bacterial cells, mammalian cells,bird cells, reptile cells, amphibious cells, microorganism cells andplant cells. Host cells may be cultured in conventional nutrient media.The media may be modified as appropriate for inducing promoters,amplifying genes or selecting transformants. The culture conditions,such as temperature, composition and pH will be apparent. Aftertransformation, transformants may be identified on the basis of aselectable phenotype. A selectable phenotype can be conferred by aselectable marker in the vector.

Transgenic Plants and Seeds

Plant cells are useful to produce tissue cultures, seeds or wholeplants. The invention includes a plant, plant part, seed, or progenythereof including a host cell transformed with a PNHX nucleic acidmolecule. The plant part is preferably a leaf, a stem, a flower, a root,a seed or a tuber.

The invention includes a transformed (transgenic) plant having increasedsalt tolerance, the transformed plant containing a nucleic acid moleculesequence encoding for Na⁺/H⁺ transporter polypeptide activity and thenucleic acid molecule sequence having been introduced into the plant bytransformation under conditions whereby the transformed plant expressesa Na⁺/H⁺ transporter in active form.

The methods and reagents for producing mature plants from cells areknown in the art. The invention includes a method of producing agenetically transformed plant which expresses PNHX or TNHX polypeptideby regenerating a genetically transformed plant from the plant cell,seed or plant part of the invention. The invention also includes thetransgenic plant produced according to the method. Alternatively, aplant may be transformed with a vector of the invention.

The invention also includes a method of preparing a plant with increasedsalt tolerance, the method comprising transforming the plant with anucleic acid molecule which encodes a TNHX transporter polypeptide, aPNHX transporter polypeptide or a polypeptide encoding a Na⁺/H⁺transporter polypeptide capable of increasing salt tolerance in a cell,and recovering the transformed plant with increased salt tolerance. Theinvention also includes a method of preparing a plant with increasedsalt tolerance, the method comprising transforming a plant cell with anucleic acid molecule which encodes a TNHX transporter polypeptide, aPNHX transporter polypeptide or a polypeptide encoding a Na+/H+transporter polypeptide capable of increasing salt tolerance in a cell,and producing the transformed plant with increased salt tolerance.

Overexpression of Na⁺/H⁺ exchangers leads to an improved ability of thetransgenic plants to uptake more monovalent cations from the growthmedia (soil) leading to an increased or enhanced tissue expansion. FIG.7 shows that transformed plants have grown larger even where no NaCl isadded to soil. Therefore, the invention also relates to methods ofproducing or growing plants with increased tissue expansion (this couldbe manifested as enhanced size, growth or growth potential and mayappear as increased or enhanced root, crown, shoot, stem, leaf, flowersize or abundance in comparison to a wild type plant). The methods ofpreparing plants that have increased tissue expansion are the same asthe methods for preparing a plant with increased salt tolerancedescribed in this application (or the methods are easily adapted, to theextent that there is a difference in the methods).

The plants whose cells may be transformed with a nucleic acid moleculeof this invention and used to produce transgenic plants include, but arenot limited to the following:

Target Plants:

Group I (Transformable Preferably via Agrobacterium Tumefaciens)

-   Arabidopsis-   Potato-   Tomato-   Brassica-   Cotton-   Sunflower-   Strawberry-   Spinach-   Lettuce-   Rice    Group II (Transformable Preferably via Biolistic Particle Delivery    Systems (Particle Bombardment)-   Soybean-   Rice-   Corn-   Wheat-   Rye-   Barley-   Atriplex-   Salicornia

The nucleic acid molecule may also be used with other plants such asoat, barley, hops, sorgum, alfalfa, sunflower, alfalfa, beet, pepper,tobacco, melon, squash, pea, cacao, hemp, coffee plants and grape vines.Trees may also be transformed with the nucleic acid molecule. Such treesinclude, but are not limited to maple, birch, pine, oak and poplar.Decorative flowering plants such as carnations and roses may also betransformed with the nucleic acid molecule of the invention. Plantsbearing nuts such as peanuts may also be transformed with the salttolerance nucleic acid molecule. A list of preferable plants is in Table5.

In a preferred embodiment of the invention, plant tissue cells orcultures which demonstrate salt tolerance are selected and plants whichare salt tolerant are regenerated from these cultures. Methods ofregeneration will be apparent to those skilled in the art (see Examplesbelow, also). These plants may be reproduced, for example by crosspollination with a plant that is salt tolerant or a plant that is notsalt tolerant. If the plants are self-pollinated, homozygous salttolerant progeny may be identified from the seeds of these plants, forexample by growing the seeds in a saline environment, using geneticmarkers or using an assay for salt tolerance. Seeds obtained from themature plants resulting from these crossings may be planted, grown tosexual maturity and cross-pollinated or self-pollinated.

The nucleic acid molecule is also incorporated in some plant species bybreeding methods such as back crossing to create plants homozygous forthe salt resistance nucleic acid molecule.

A plant line homozygous for the salt tolerance nucleic acid molecule maybe used as either a male or female parent in a cross with a plant linelacking the salt tolerance nucleic acid molecule to produce a hybridplant line which is uniformly heterozygous for the nucleic acidmolecule. Crosses between plant lines homozygous for the salt resistancenucleic acid molecule are used to generate hybrid seed homozygous forthe resistance nucleic acid molecule.

The nucleic acid molecule of the invention may also be used as a markerin transformation experiments with plants. A salt sensitive plant may betransformed with a salt tolerance nucleic acid molecule and a nucleicacid molecule of interest which are linked. Plants transformed with thenucleic acid molecule of interest will display improved growth in asaline environment relative to the non-transformed plants.

Fragments/Probes

Preferable fragments (fragments are also referred to as polypeptidefragments or peptide fragments) include 10 to 50, 50 to 100, 100 to 250,250 to 500, 500 to 1000, 1000 to 1500, or 1500 or more nucleotides of anucleic acid molecule of the invention. A fragment may be generated byremoving a single nucleotide from a sequence in FIG. 1 or 5 (or apartial sequence thereof). Fragments may or may not have Na⁺/H⁺transporter activity.

The nucleic acid molecules of the invention (including a fragment of asequence in FIG. 1 or 5 (or a partial sequence thereof) (such as SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7) can be used as probes todetect nucleic acid molecules according to techniques known in the art(for example, see U.S. Pat. Nos. 5,792,851 and 5,851,788). The probesmay be used to detect nucleic acid molecules that encode polypeptidessimilar to the polypeptides of the invention. For example, a probehaving at least about 10 bases will hybridize to similar sequences understringent hybridization conditions (Sambrook et al. 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor).

The invention includes oligonucleotide probes made from the AtNHXsequences described in this application or other nucleotide sequences ofthe invention. The probes may be about 10 to 30 or 15 to 30 nucleotidesin length and are preferably at least 30 or more nucleotides. Apreferred probe is 5′-TTCTTCATATATCTTTTGCCACCC-3′ SEQ ID NO:36 (codingfor the amiloride binding domain) or at least about 10 or 15 nucleotidesof this sequence. The invention also includes an oligonucleotideincluding at least 30 consecutive nucleotides of an AtNHX molecule inFIG. 1 or 5 (or a partial sequence thereof). The probes are useful toidentify nucleic acids encoding AtNHX, polypeptides and proteins otherthan those described in the application, as well as peptides,polypeptides, and proteins have Na⁺/H⁺ transporter activity andpreferably functionally equivalent to AtNHX. The oligonucleotide probesare capable of hybridizing to one or more of the sequences shown in FIG.1 or 5 (or a partial sequence thereof) or the other sequences of theinvention under low, moderate or high stringency hybridizationconditions. A nucleotide sequence encoding a polypeptide of theinvention may be isolated from other organisms by screening a libraryunder low, moderate or high stringency hybridization conditions with adetectable probe (e.g. a labeled probe). The activity of the polypeptideencoded by the nucleotide sequence may be assessed by cloning andexpression of the DNA. After the expression product is isolated, thepolypeptide is assayed for Na⁺/H⁺ transporter activity as described inthis application.

Functionally equivalent AtNHX, TNHX or PNHX nucleic acid molecules fromother cells, or equivalent AtNHX, TNHX or PNHX-encoding cDNAs orsynthetic DNAs, can also be isolated by amplification using PolymeraseChain Reaction (PCR) methods. Oligonucleotide primers, includingdegenerate primers, based on the amino acid sequence of the sequences inFIGS. 1 or 5 (or a partial sequence therof) can be prepared and used inconjunction with PCR technology employing reverse transcriptase toamplify functionally equivalent DNAs from genomic or cDNA libraries ofother organisms. Alternatively, the oligonucleotides, includingdegenerate nucleotides, can be used as probes to screen cDNA libraries.

Thus, the invention includes an oligonucleotide probe comprising all orpart of a nucleic acid in FIG. 1 or 5 (or a partial sequence thereof),or a complementary strand thereof. The probe is preferably labeled witha detectable marker. The invention also includes an oligonucleotidecomprising at least 10, 15 or 30 nucleotides capable of specificallyhybridizing with a sequence of nucleic acids of the nucleotide sequenceset forth in FIG. 1 or 5 (or a partial sequence thereof). The inventionalso includes a single strand DNA primer for amplification of PNHXnucleic acid, wherein the primer is selected from a nucleic acidsequence derived from a nucleic acid sequence in FIG. 1 or 5 (or apartial sequence thereof).

The invention also includes a method for identifying nucleic acidmolecules encoding a TNHX, PNHX or AtNHX polypeptide. Techniques forperforming the methods are described in, for example, U.S. Pat. Nos.5,851,788 and 5,858,719. A preferred method includes contacting a samplecontaining nucleic acids with an oligonucleotide, wherein saidcontacting is effected under low, moderate or high stringencyhybridization conditions, and identifying nucleic acids which hybridizethereto. Hybridization forms a hybridization complex. The presence of acomplex correlates with the presence of a nucleic acid molecule encodingTNHX, plant PNHX polypeptide or AtNHX in the sample. In a preferredmethod, the nucleic acid molecules are amplified by the polymerase chainreaction prior to hybridization.

Kits

The invention also includes a kit for conferring increased salttolerance to a plant or a host cell including a nucleic acid molecule ofthe invention (preferably in a composition fo the invention) andpreferably reagents for transforming the plant or host cell.

The invention also includes a kit for detecting the presence of a TNHXor a PNHX nucleic acid molecule, comprising at least one oligonucleotideof the invention. Kits may be prepared according to known techniques,for example, see U.S. Pat. Nos. 5,851,788 and 5,750,653.

Antibodies

The invention includes an isolated antibody immunoreactive with apolypeptide of the invention (see Example 1). The antibody may belabeled with a detectable marker or unlabeled. The antibody ispreferably a monoclonal antibody or a polyclonal antibody. TNHX, PNHX orAtNHX antibodies can be employed to screen organisms containing TNHX,PNHX or AtNHX polypeptides. The antibodies are also valuable forimmuno-purification of polypeptides from crude extracts.

The isolated antibody is preferably specifically reactive with a TNHX orPNHX transporter, preferably an AtNHX transporter. The transporter ispreferably encoded by a nucleic acid molecule in FIG. 1 (or moleculesthat hybridize to a molecule in FIG. 1 under low, moderate or highstringency hybridization conditions or molecules having at least about:17%, at least 20%, at least 25%, or at least 35% sequence identity (orthe other preferred percentages of identity or sequence similaritydescribed above) to a molecule in FIG. 1 or 5 (or a partial sequencethereof). The transporter is preferably a polypeptide in FIG. 1 (orpolypeptides having at least about: 28%, 35% sequence identity (or theother preferred percentages of identity or sequence similarity describedabove) to a polypeptide in FIG. 1 or 5 (or a partial sequence thereof.The antibody preferably does not cross-react with other transporterpolypeptides. The antibody is preferably specifically reactive with apolypeptide having an amino acid sequence encoded by a nucleic acidmolecule set forth in FIG. 1 or 5 (or a partial sequence thereof.

Examples of the preparation and use of antibodies are provided in U.S.Pat. Nos. 5,792,851 and 5,759,788. For other examples of methods of thepreparation and uses of monoclonal antibodies, see U.S. Pat. Nos.5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356, 5,591,628,5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705. Examples ofthe preparation and uses of polyclonal antibodies are disclosed in U.S.Pat. Nos. 5,512,282, 4,828,985, 5,225,331 and 5,124,147.

The invention also includes methods of using the antibodies. Forexample, the invention includes a method for detecting the presence ofTNHX, PNHX or AtNHX transporter polypeptide, by: a) contacting a samplecontaining one or more polypeptides with an antibody of the inventionunder conditions suitable for the binding of the antibody topolypeptides with which it is specifically reactive; b) separatingunbound polypeptides from the antibody; and c) detecting antibody whichremains bound to one or more of the polypeptides in the sample.

Research Tool

Cell cultures, seeds, plants and plant parts transformed with a nucleicacid molecule of the invention are useful as research tools. Forexample, one may obtain a plant cell (or a cell line, such as animmortalized cell culture or a primary cell culture) that does notexpress AtNHX1, insert an AtNHX1 nucleic acid molecule in the cell, andassess the level of AtNHX1 expression and activity. Alternatively, PNHXnucleic acid molecules may be overexpressed in a plant that expresses aPNHX nucleic acid molecule. In another example, experimental groups ofplants may be transformed with vectors containing different types ofPNHX nucleic acid molecules (or PNHX nucleic acid molecules similar toPNHX or fragments of PNHX nucleic acid molecules) to assess the levelsof protein produced, its functionality and the phenotype of the plants(for example, phenotype in saline soil). The polypeptides are alsouseful for in vitro analysis of TNHX, PNHX or AtNHX activity orstructure. For example, the polypeptides produced can be used formicroscopy or X-ray crystallography studies.

The TNHX, PNHX or AtNHX nucleic acid molecules and polypeptides are alsouseful in assays. Assays are useful for identification and developmentof compounds to inhibit and/or enhance polypeptide function directly.For example, they are useful in an assay for evaluating whether testcompounds are capable of acting as antagonists for PNHX polypeptides by:(a) culturing cells containing: a nucleic acid molecule which expressesPNHX polypeptides (or polypeptides having PNHX or Na+/H+ activity)wherein the culturing is carried out in the presence of: increasingconcentrations of at least one test compound whose ability to inhibittransport activity of PNHX polypeptide is sought to be determined, and afixed concentration of salt; and (b) monitoring in the cells the levelof salt transported out of the cytosol as a function of theconcentration of the test compound, thereby indicating the ability ofthe test compound to inhibit PNHX transporter activity. Alternatively,the concentration of the test compound may be fixed and theconcentration of salt may be increased.

Another experiment is an assay for evaluating whether test compounds arecapable of acting as agonists for PNHX polypeptide characterized bybeing able to transport salt across a membrane, (or polypeptides havingPNHX or Na+/H+ transporter activity) by (a) culturing cells containing:a nucleic acid molecule which expresses PNHX polypeptide or (orpolypeptides having PNHX activity) thereof, wherein said culturing iscarried out in the presence of: fixed concentrations of at least onetest compound whose ability to increase or enhance salt transportactivity of PNHX polypeptide is sought to be determined, and anincreasing concentration of salt; and (b) monitoring in the cells thelevel of salt transported out of the cytosol as a function of theconcentration of the test compound, thereby indicating the ability ofthe test compound compound to increase or enhance PNHX polypeptideactivity. Alternatively, the concentration of the test compound may befixed and the concentration of salt may be increased. Suitable assaysmay be adapted from, for example, U.S. Pat. No. 5,851,788. It isapparent that TNHX and AtNHX may also be used in assays.

Bioremediation

Soils containing excessive salt may be unable to grow plants in a mannersuitable for agriculture. The invention includes a method for removingsalt from a growth medium, comprising growing a plant transformed with anucleic acid molecule of the invention and expressing a salt toleranceNa⁺/H⁺ transporter polypeptide in the growth medium for a time periodsufficient for the plant root to uptake and accumulate salt in the rootor shoot biomass. The growth medium may be a solid medium, semi-solidmedium, liquid medium or a combination thereof. It may include soil,sand, sludge, compost, or artificial soil mix. The shoot (leaf or stem)or and root biomass may be harvested. Preferably, a sufficient portionof the shoot biomass is not harvested and is left in the growth media topermit continued plant growth.

Using Exogenous Agents in Combination with a Vector

The nucleic acid molecules of the invention may be used with othernucleic acid molecules that relate to salt tolerance, for example,osmoregulant genes. Host cells or plants may be transformed with thesenucleic acid molecules. Osmoregulants are disclosed, for example, inU.S. Pat. Nos. 5,563,324 and 5,639,950.

It will be clear to those skilled in the art that sequences in FIGS. 1(c) and 5(a) and (b) are also useful, for example in preparation ofprobes or as experimental tools or as antigens to which antibodies maybe directed. The following Examples are intended to illustrate andassist in the further understanding of the invention. Particularmaterials employed, species, conditions and the like are not intended tolimit the reasonable scope of the invention.

EXAMPLE 1

Preparation of Polyclonal and Monoclonal Antibodies.

Hydropathy profiles of the Arabidopsis Na⁺/H⁺ antiport revealed arelatively hydrophilic domain (at the C-terminus) with possibleregulatory functions. The C-terminus was sub-cloned into the pGEX-2TKvector (Pharmacia) to allow the overexpression of the C-terminuspolypeptide as a GST-fusion polypeptide in E. coli. The fusionpolypeptide was purified by glutathione-affinity chromatography and usedas an antigen in rabbits to obtain polyclonal antibodies (30).

Monoclonal antibodies are prepared in mice hybridomas according toestablished techniques (30) using the C-terminus polypeptide asdescribed above. Polyclonal and monoclonal antibodies raised againstother regulatory regions of the Arabidopsis Na⁺/H⁺ antiport are alsoobtained as described above. The invention includes the antibodies andthe hybridoma which secretes the monoclonal antibodies.

EXAMPLE 2

Identification of Homologous Nucleic Acid Molecules from Other PlantSpecies, Preferably Salt Tolerant Species.

Several experimental approaches are used to identify homologous nucleicacid molecules from salt tolerant species. a) We screen cDNA and genomiclibraires from sugar beets (a moderate salt-tolerant crop, also known asred beet) under low-stringency conditions with an Arabidopsis Na⁺/H⁺antiport cDNA as a probe (31); b) We apply PCR techniques usingdegenerate oligonucleotide primers designed according to the conservedregions of the Arabidopsis Na⁺/H⁺ antiport (32); c) We screen cDNAexpression libraries from different plants (salt-tolerant andsalt-sensitive) using antibodies raised against an Arabidopsis Na⁺/H⁺antiport (31). We also use bioinformatics techniques to identify nucleicacid molecules. The invention includes methods of using such a nucleicacid molecule, for example to express a recombinant polypeptide in atransformed cell.

The techniques described above for isolating nucleic acid molecules fromArabidopsis and sugar beet are used to isolate a salt tolerance nucleicacid molecule from Atriplex and other plants.

EXAMPLE 3

Overexpression of the PNHX Transporter, Preferably ArabidopsisTransporter (AtNHX).

The Na+/H+ antiport is expressed in Arabidopsis plants, although thewild type plants show impaired growth at NaCl concentrations higher than75 mM. The Na+/H+ antiport is overexpressed in these plants in order toimprove their tolerance to high salt concentrations. A full length cDNA(preferably coding for the AtNHX1 polypeptide (AtNHX3, AtNHX4 or AtNHX5)cloned from an Arabidopsis thaliana (Columbia) seedling cDNA library isligated into a pBINSl vector (33). This vector contains a constitutivelystrong promotor (“super-promotor” (20)). Also, T-DNA vectors (pBECKS)are used (34). Constructs containing the AtNHX1 cDNA with the fullNa+/H+ antiport open reading frame in a sense orientation were selectedby colony hybridization using the AtNHXi as a probe and byrestriction-digest analysis using BgIII restriction endonuclease. Theseconstructs are used to transform Agrobacterium tumefaciens, and thesetransformed Agrobacterium tumefaciens are used for transformation ofArabidopsis plants. The Agrobacterium for inoculation is grown at 28° C.in a medium containing 5 g/l Bacto Beef Extract, 5 g/l Bacto-Peptone, 1g/l Bacto Yeast Extract, 240 mg MgSO.sub.4 and 5 g/l sucrose. The pHwill be adjusted to 7.2 with NaOH.

Arabidopsis seeds are washed and surface-sterilized in 5% (w/v) sodiumhypochlorite containing 0.15% (v/v) Tween-20. The seeds are rinsedthoroughly with sterile distilled water. Seed aliquots are dispensed inflasks containing 45 ml of cocultivation medium (MS salts, 100 mMsucrose, 10 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid,100 mg/l inositol and the pH adjusted to 6.0 with KOH. The flasks areincubated at 22° C. under constant rotation (190 rpm) and constantlight. After 10-18 h (time needed to break clumps of seeds) 5 ml of logphase of Agrobacterium (OD₆₀₀=0.75) carrying the AtNHX1 construct areadded. Twenty-four hours following the inoculation, the seeds are driedby filtration and sown into pre-soaked vermiculite. The flats containingthe seeds are irrigated as required with a half-Hoagland solution. Theflats are covered with plastic to prevent desiccation and maintained atlow artificial illumination. After 3 days the flats are transferred tothe greenhouse (the plastic cover removed) under a 16/8 day/night cycle.Supplementary light is provided by high pressure sodium vapor lights.Seven weeks after sowing, the plants are dried thoroughly and the seeds(T2) harvested. Transformation efficiency is estimated by plating100,000 seeds (approximately 2.5 g of seeds) on agar plates containing50 mg/l kanamycin in a medium containing 1% (w/v) sucrose, 0.8 (w/v)agar, MS salts and a pH 6.0 adjusted with KOH. The plates aretransferred to a growth room at 25° C. under continuous light. After 10days the kanamycin-resistant seedlings are transferred to new growthmedium for 2 weeks and then transferred to small pots containingvermiculite. At senescence (8 weeks) the seeds are collected from singleplants (T3). These seeds are germinated and used to assess salttolerance of the transgenic plants.

EXAMPLE 4

Overexpression of TNHX or PNHX in Other Plants.

In a preferred method, overexpression of PNHX, preferably AtNHX1,AtNHX3, AtNHX4 or AtNHX5, in a number of plants (potato, tomato,brassica, cotton, sunflower, strawberries, spinach, lettuce, rice,soybean, corn, wheat, rye, barley, atriplex, salicornia, and others) isachieved by Agrobacterium tumefaciens-based transformation and/orparticle bombardment AtNHX3, AtNHX4, AtNHX5 are also useful in thisexample). The full length cDNA (coding for the AtNHX1) is ligated intothe pBINS1 vector or pBECKS (as described above) and these constructsare used to transform Agrobacterium tumefaciens strain LBA4404.Agrobacterium used for inoculation is grown as described above. Culturedcells (callus), leaf explants, shoot and root cultures are used astargets for transformation. The targeted tissues are co-cultivated withthe bacteria for 1-2 days. Afterwards, the tissue is transferred to agrowth media containing kanamycin. After one week the tissue istransferred to a regeneration medium containing MS salts, 1% sucrose,2.5 mg/l 3-benzyladenine, 1 mg/l zeatin, 0.75% agar and kanamycin.Weekly transfers to fresh regeneration media are performed.

In another preferred embodiment, overexpression constructs carrying theAtNHX1 cDNA are introduced into an electro-competent Agrobacteriumiumefaciens (LBA4404) by electroporation. The Agrobacteria are plated onLB plates containing 50 mg/L kanamycin and grown for ˜2 days at 30° C.2to select for bacteria carrying the overexpression constructs. One literliquid LB+kanamycin (50 mg/L) is inoculated with a single Agrobacteriumcolony selected from the LB (kanamycin 50 mg/L) plates. The culture isgrown to a minimum of OD=1 (600 nm) for 2-3 days. The Agrobacteria arethen pelleted and resuspended in 1 L infiltration medium (IM-0.5X MSsalts; 0.5 g/L MES; 5% sucrose; 0.03% Silwet L-77). FloweringArabidopsis plants with primary bolts reaching ˜15 cm are used for thetransformation procedure (T1). Pots of Arabidopsis plants are dunkedinto the 1 M solution containing the Agrobacteria and left submerged for2-6 minutes. The same procedure can be repeated after 8-12 days on thesame plants. Plants are allowed to senesce, the plants are driedthoroughly and the seeds harvested. Seeds are plated on agar platescontaining 25 mg/L kanarnycin in a medium containing MS salts, 0.8%(w/v) agar adjusted to pH 6.0 with KOH. The plates are transferred to agrowth room at 25° C. under continuous light. After 10 days thekanamycin-resistant seedlings (T2) are transferred to small potscontaining vermiculite. At senescence (˜8 weeks) the seeds are collectedfrom single plants and plated on agar plates containing MS salts and 25mg/L kanamycin. After 10 days the kanamycin-resistant seedlings (T3) aretransferred to small pots containing vermiculite. Seeds produced bythese plants are germinated and used to assess salt tolerance of thetransgenic plants. A biolistic particle delivery system (particlebombardment) is also used for the overexpression of NHX (AtNHX1, AtNHX3, AtNHX4 or AtNHX5 are useful for this example). Constructs made using aplasmid vector preferably carrying a constitutive promoter, the AtNHX1open reading frame in a sense orientation and a NOS termination site areused. Plasmid DNA is precipitated into 1.25 mg of 1-2 μm gold particlesusing 25 μl of 2.5 M CaCl₂ and 10 μl of 0.1 M thiamine (free base).DNA-coated particles are washed with 125 μl of 100% ethanol and thenresuspended in 301 μl ethanol. The samples are sonicated to obtain anefficient dispersion, and the samples are aliquoted to obtain deliverydisks containing 3 μg of DNA each. Particle bombardment is optimizedaccording to the specific tissue to be transformed. Tissue samples areplaced in Petri dishes containing 4.5 g/l basal MS salts, 1 mg/lthiamine, 10 mg/l myoinositol, 30 g/l sucrose, 2.5 mg/l amphotericin and10 mM K₂HPO₄ at pH 5.7. After bombardment the petri dishes are incubatedfor 18-24 hours. Tissue is regenerated in plates with growth mediacontaining the selective marker. Rooting is initiated and transformedplants are grown under optimal growth conditions in growth chambers.After 2-4 weeks the seedlings are transferred to new growth medium for 2weeks and then transferred to small pots containing vermiculite. Atsenescence the seeds are collected from single plants. These seeds aregerminated and used to assess salt tolerance of the transgenic plants.

EXAMPLE 5

Overexpression of AtNHX1-Homologs in Other Plants.

Overexpression of AtNHX1-homologs from other plant species, preferablysalt tolerant species (i.e., sugar beet) in other plants (potato,tomato, brassica, cotton, sunflower, strawberries, spinach, lettuce,rice, soybean, corn, wheat, rye, barley, atriplex, salicornia, andothers) is achieved by Agrobacterium tumefaciens-based transformationand/or particle bombardment as described above (in Examples 3 and 4).Regeneration of the transformed plants is performed as described inExamples 3 and 4 (AtNHX3, AtNHX4 or AtNHX5).

EXAMPLE 6

Expression of PNHX, AtNHX1, AtNHX1 Homologs and AtNHX1 Derivatives inSaccharomyces cerevisiae.

Expression of TNHX or PNHX, preferably AtNHX1, AtNHX1 homologs (such asAtNHX3, AtNHX4, AtNHX5), and AtNHX1 derivatives in yeast is useful toassess and characterize the biochemical properties of the recombinantand native polypeptides. Expression in yeast also facilitates the studyof interactions between AtNHX1, its homologs and derivatives withregulatory polypeptides. We have made conditional expression constructsby ligating the coding region of the AtNHX1 cDNA into two vectors, pYES2(Invitrogen) and pYEP434 (35). Both constructs providegalactose-inducible expression, but pYES2 has a URA3 selectable markerwhile pYEP434 has LEU2 as a selectable marker. Transformation by lithiumacetate (36), 1994), is followed by selection on solid media containingamino acids appropriate for the selection of cells containing thetransformation vector. For integrative transformation, the YXplac seriesof vectors for integrative transformation are used (37).

EXAMPLE 7

Molecular Characterization and Functional Analysis of Na⁺/H⁺ Exchangersfrom Arabidopsis and Other Plants, Preferably Salt-Tolerant (Halophytes)Plants.

We do molecular and biochemical characterization of the different Na⁺/H⁺exchangers from Arabidopsis and other plants, preferably salt tolerantplants (halophytes). We determine the expression patterns of thedifferent Arabidopsis putative exchangers. Using Northern blot analysiswith isoform-specific cDNA probes under high stringency conditions andstandard molecular biology protocols, we determine thetissue-specificity, developmental and salt-inducibility gene expressionprofiles of each isoform.

We employ common molecular biology procedures to isolate Na⁺/H⁺exchangers from other plants (Table 5), in particular halophytes (suchas Beta vulgaris, Atriplex, Messembryanthemum chrystalinum, etc.). Wedesigned degenerate oligonucleotide PCR primers, based upon highlyconserved regions within Na⁺/H⁺ exchangers (one within theamiloride-binding domain, and another within a region about 200 aminoacid residues further downstream) from Arabidopsis, yeast, mammals, andC. elegans, to generate a 600-1,000 bp DNA fragments by PCR. Sequencingof these products revealed significant homology to AtNHX1 and they aretherefore being used as a probe to screen the different halophyte cDNAlibraries to isolate the full-length cDNAs by standard methods. We usethe nucleic acid molecules obtained in this procedure in methods ofproducing transgenic host cells and plants as described above.

We have subcloned unique regions from AtNHX1, AtNHX3 and AtNHX5 isoformsinto a prokaryotic expression vector (pGEX2TK, Pharmacia) for theproduction of recombinant GST-fusion proteins that are being used forthe generation of isoform-specific polyclonal antibodies in rabbits.Briefly, sequence-specific oligonucleotides, with 5′ BamHI (sensestrand) and 3′ EcoRI (antisense strand) flanking restriction sites, wereused for PCR-mediated amplification of the unique (partial) codingregions from each isoform, and the digested PCR products were ligatedinto EcoRI/BamHI-digested pGEX2TK vector. pGEX2TK plasmids containingthe inserts corresponding to each AtNHX isoform were sequenced on bothstrands to verify the fidelity of the PCR reaction and were used forexpression and purification of the recombinant GST-fusion proteins in E.coli (BL21 pLysS) as per manufacturers instructions (Pharmacia). Wefollow an identical procedure to that described above to producerecombinant halophyte-PNHX GST-fusion protein in E. coli. Antibodiesagainst the fusion proteins are produced in rabbits by standardprocedures and their isoform-specificity are confirmed by westernblotting using the different GST-fusion proteins. The antibodies areused in conjunction with subcellular membrane fractions (prepared fromsucrose density gradients) (15) from various Arabidopsis and other planttissues, preferably halophyte tissues and western blots to determine thesubcellular localization of each Na+/H+ exchanger isoform. Theselocalization studies assign functions to the various isoforms.

EXAMPLE 8

Biochemical Characterization and Functional Analysis of Na⁺/H⁺Exchangers from Arabidopsis and Other Plants, Preferably Salt-Tolerant(Halophytes) Plants.

Biochemical characterization of the Na⁺/H⁺ exchanger isoforms isperformed in (i) heterologous eukaryotic expression systems (baculovirusexpression system in Sf9 insect cells, transgenic yeast); and in (ii)transgenic plants.

The use of heterologous expression systems allows the fastcharacterization of the kinetic properties of each exchanger isoform(K_(m), V_(max), ion specificity). Baculovirus-infected Sf9 cells haveproven to be a useful and adaptable system for high-level expression ofcorrectly folded eukaryotic membrane proteins, thus they are an idealtool for the study of membrane-bound proteins. The large size of thecells, combined with the relatively short time needed for the expressionof the foreign plasma membrane-bound proteins (3-4 days) provides anexcellent experimental system for the application of isotope exchangetechniques. For expression in Sf9 insect cells, the Invitrogenbaculovirus Sf9 insect cell system is used. Expression vector constructs(pBluBac4.5, Invitrogen) encoding full-length AtNHX exchanger proteinsare prepared for each AtNHX and other PNHX isoforms using a PCR-basedsubcloning approach similar to that described above for the generationof GST-fusion proteins. Initially, the suitability of the insect cellexpression system for uptake analysis is performed using a single AtNHXisoform. The other PNHX isoforms are studied in a similar manner.Cultures of Sf9 insect cells are infected with baculovirus containingexpression vector constructs encoding the different PNHX isoforms.Infection and selection of transformants are performed as permanufacturer's instructions (Invitrogen). The isoform-specificantibodies described above aid in the assessment of recombinant proteinexpression and localization within the insect cells.

Equally important is the use of transgenic yeast as a tool for theexpression of recombinant eukaryotic proteins, particularly because ofpost-translational modifications and targeting to endomembranes. Inaddition, functional complementation of yeast mutant strains with plantproteins is often possible. We have subcloned the AtNHX1 cDNA into ayeast expression vector (pYES2) using a PCR-based approach as describedabove. Yeast (strain w303a) have been transformed with this constructand expression of the recombinant plant protein is confirmed once theantiserum is available. In addition, salt-tolerance of transformed yeastisassessed for each AtNHX isoform by comparing growth rates at differentNaCl concentrations. Methods for the isolation of transport-competentplasma membranes and tonoplast and the isolation of intact vacuoles areperformed. The kinetics of H⁺/Na⁺ exchange is measured in intact insectcells and yeast, intact yeast vacuoles, and isolated plasma membranesand tonoplast vesicles according to known methods. Na⁺ influx in intactcells is monitored by isotopic exchange using (²²Na⁺)Cl andfast-filtration techniques (17,i,ii). Kinetics of H⁺-dependent Na⁺fluxes in vesicles is monitored by following the pH-dependentfluorescent quench of acridine dyes (13,17).

The results of these kinetic characterization studies providesinformation about the ion specificity, affinity, and optimal activityconditions for each AtNHX isoform. We assign the activity of eachisoform to the corresponding target membrane, and we also determinewhich of the isoforms have a higher affinity for sodium. We characterizethe mechanisms of salt tolerance in general and tissue-specificity anddevelopmental expression in particular.

In transgenic plants, expression of the different Na⁺/H⁺ antiports isverified with western blots using the isoform-specific antibodiesdescribed above. The kinetics of H⁺/Na⁺ exchange is measured in intactvacuoles, isolated plasma membranes and tonoplast vesicles (from rootsand leaves) as described above.

EXAMPLE 9

Identification of Positive and Negative Regulators of Na⁺/H⁺ AntiportActivity.

Heterologous expression of plant transport molecules in Saccharomycescerevisiae has been used successfully in recent years in numerousstudies. The availability of yeast mutants with salt-sensitivephenotypes (generated by ‘knock-outs’ of sodium transport molecules suchas Δena1-4-the plasma membrane Na⁺-ATPase pumps) makes it an especiallysuitable system for the study of sodium transport molecules. Thisheterologous expression facilitates kinetic studies of the antiportactivity in yeast cells using radiolabelled ²²Na⁺.

Successful suppression of yeast mutants, incapable of sodiumdetoxification allows for the genetic identification of positive andnegative regulators of these Na⁺/H⁺ antiports. Mutant yeast cells havinga suppressed phenotype as a result of the expression of a plant Na⁺/H⁺antiport are transformed with an Arabidopsis cDNA library for thepurpose of identifying particular regulators of these antiportmolecules. A phenotype of increased sodium tolerance in yeast identifiesparticular positive regulators of the antiport activity while negativeregulators are identified by a phenotype of decreased sodium tolerance.These phenotypes depend on the co-expression of the particular cDNAsidentified along with that of the Na⁺/H⁺ antiport under investigation.Identification of essential amino acid residues regulating the activityof Na⁺/H⁺ exchanger molecules is investigated by random mutagenesis ofthe antiport molecule which is achieved by PCR using a commerciallyavailable low fidelity Taq enzyme. The constructs generated are used intransforming sodium-related yeast mutants to identify particular Na⁺/H⁺antiport residues that affect suppression of the mutant yeast phenotype.Both gain-of-function and loss-of-function mutations are examined andmapped to the particular mutant residue by sequencing. Gain-of-functionmutations are of particular interest since they represent constitutiveactivation of the antiport activity allowing for increased sodiumdetoxification.

EXAMPLE 10

Transformation of Arabidopsis thaliana Using Overexpression of DifferentPutative Isoforms and Antiports from other Plants Preferably SaltTolerant Plants and Evaluation of Salt-Tolerance.

Arabidopsis represents a readily transformable model organism with theparticular advantage of having a short generation time. Agrobacteriumtumefaciens-mediated genetic transformation is utilized for Arabidopsis(ecotype Columbia). Studies include the overexpression of PNHXtransgenes in a wild-type background, combined overexpression of morethan one PNHX transgene, and suppression of endogenous PNHX expressionusing antisense PNHX expression. Stable transformation of progeny isconfirmed by Southern blotting. Overexpression of transgenes, orsuppression of expression using antisense constructs, is confirmed byNorthern and western blotting. In all cases, salt-tolerance oftransgenic plants is compared to wild-type plants, and control plantstransformed with empty transformation vectors. Separate transformationsare performed on Arabidopsis plants using expression vector constructsfor each of the different AtNHX isoforms. In addition, Arabidopsisplants are transformed with PNHX genes from other plants, preferablysalt tolerant plants in order to assess the effect on salt tolerance ofthe expression of a Na⁺/H⁺ exchanger in a glycophytic plant.

For overexpression studies, full-length AtNHX1, AtNHX3, AtNHX4 andAtNHX5 cDNAs are subcloned in a sense orientation into the expressionvector containing a “superpromoter” (20). A PCR based subcloningstrategy is used for each AtNHX cDNA as described above for theproduction of NHXGST-fusion constructs. For the production of vectorconstructs containing PNHX cDNAs in an antisense orientation,oligonucleotides with SalI and SacI restriction sites flanking theC-terminal and N-terminal PNHX regions respectively, are used for PCRamplification. All plasmid constructs are sequenced on both strands toconfirm the fidelity of the PCR amplification before transformation ofAgrobacterium tumefaciens (strain LBA4404). For each PNHX-pBISN1construct, approximately 1 L of Agrobacterium culture, grown underantibiotic selection at 28° C., is used for the transformation ofArabidopsis. Plants are ready for transformation when primary bolts areapproximately 15 cm. About 2 flats of plants (−80 plants per flat) areused per transformation. A highly efficient, vacuum-less infiltrationtransformation method (iii) is used. Harvested Agrobacterium culturesare resuspended in an infiltration media containing a mild surfactant(Silwet L-77, Lehle Seeds), and each pot of Arabidopsis is simplysubmerged in the Agrobacterium for 2-6 minutes. Plants are thereafterdrained, and returned to the growth chamber until the seeds are readyfor harvesting (about 4 weeks). Seeds (T1 generation) are collected andafter surface sterilization, are plated on sterile, selective mediacontaining kanamycin, vernalized, and then grown under optimalconditions. Healthy seedlings showing kanamycin resistence after about 7days are transplanted to soil and the presence of the transgeneconfirmed by Southern blotting. Seeds from T1 transformants (ie T2generation) are harvested, sown, and T2 plants used for Northern andwestern blotting to determine the expression patterns of the transgenesand PNHX proteins. Representative transgenic lines (e.g. showing low,medium, or high transgene expression) is used for studies ofsalt-tolerance. A similar approach is used for transformation ofArabidopsis with the PNHXs from other plants.

Salt tolerance is assessed by measuring the growth rate of the plants atincreasing salt concentrations. Plant biomass, root/shoot ratios, tissueion content is measured. Root and hypocotyl growth rates is measured andcorrelated with tissue water content of plants growing at different NaClconcentrations.

EXAMPLE 11

Transformation of Crop Plants with A. Thaliana and/or Other exchangersunder constitutive and Inducible Promoters and Evaluation ofSalt-Tolerance.

-   a) Agrobacterium tumefaciens-mediated transformation of crop plants

We assess whether or not homologues of the AtNHX genes exist in theplant of choice. We use degenerate oligonucleotide PCR-primers (asdescribed for other plants) and a cDNA library to isolate thefull-length cDNA. The high efficiency Agrobacterium-mediatedtransformation method developed specifically for Brassica by Moloney etal (iv) is used to introduce and overexpress foreign nucleic acidmolecules and/or overexpress the endogenous PNHX nucleic acid moleculein the crop plant(s). This method takes advantage of the fact that cutcotyledonary petioles from, which are capable of undergoingorganogenesis (ie generating explants), are very susceptible toAgrobacterium infection. Shortly after germination (˜5 days) cotlyedonsare excised and imbedded into Murashige-Skoog medium (Gibco) enrichedwith benyzladenine. Expression vector constructs are prepared using aPCR-based subcloning approach as described above using the pCGN5059binary plasmid (which employs the CaMV ³⁵S promoter to driveconstitutively high expression) engineered for gentamycin resistance(iv) and cDNAs of the various AtNHX clones and/or the halophyte PNHXclones, and the choosen plant PNHX clones. Excised cotyledons areinfected with Agrobacterium cultures (strain EHA101), containing thevector construct of interest, by brief dipping and then co-cultivatedwith the Agrobacterium for a 72 h. Subsequently, cotlyedons aretransferred to regeneration medium containing gentamycin as theselective agent. After explant regeneration, and subculturing, onselective media (˜4 weeks) explants are transferred to rooting mediumand then into soil once a root mass has developed. Tissue samples areexamined from growing plants to confirm transgene presence by Southernblotting as described above for the transformation of Arabidopsis.Transformed plants (T1 generation) are allowed to flower and set seedand these seeds are germinated (T2) under selective conditions andtransformants used for expression analysis of the transgenes andevaluation of salt-tolerance as described above. Also, biochemicalanalysis of the plants is performed. These include, Na⁺/K⁺ ratios,sugar, amino acid and quaternary N-compounds. Salt-tolerance is alsoevaluated in fields trials.

-   b) Microprojectile Bombardment-Mediated Transformation of Crop    Plants.

A microprojectile bombardment-mediated transformation of crop plants isused when Agrobacterium tumefaciens-mediated transformation is notsuccessful. We assess whether or not homologues of the AtNHX genes existin the plant of choice. We use degenerate oligonucleotide PCR-primers(as described above) and a cDNA library to isolate the full-length cDNA.Expression vector constructs, using the pBAR vector for high levelexpression of AtNHX or the halophyte PNHX or the endogenous PNHX fromthe plant of choice, are used in conjunction with the microprojectilebombardment system as described by Tomes et al. (v). Bombardmentprocedures is carried out in callus tissue. Plant calli are initiated byculturing immature embryos on Callus medium (vi). After about 2 weeks,friable calli that are growing rapidly are subcultured and grown for anadditional 2 weeks and then used for transformation. Calli fortransformation are transferred to fresh medium, incubated for 24 h andbombarded with tungsten microprojectiles carrying the pBARNHX vectorconstruct. Bombardment conditions is performed according tomanufacturer's instructions. Calli that show visible growth 10 daysafter bombardment are transferred to selective media (containing eitherBialaphos or Ignite) in order to identify putative transformants. Thegrowth of transformed plant calli on this selective media is continuedfor 3-4 months. Each putative stable transgenic event becomes apparentas a mass of friable embyogenic callus growing in the presence of theselection agent. Stable transformation is verified by Southern blots.Selected calli are transferred onto a regeneration medium (v), kept inthe dark at 28° C. for 7 days and then transferred to growth chambersunder a 16-h photoperiod until green shoots appear. Plantlets (1-2 cmlong) are transferred to individual tubes containing germination mediumto allow continued development. At the three to four leaf stage, plantsare transferred to soil and into the greenhouse. At the eight-leafstage, these plants are sprayed with 1% (w/v) Ignite herbicide to detectthe presence of the BAR gene. This herbicide kills those plants notcarrying the BAR gene. Confirmed transgenic plants (T1) are allowed tomature, flower, set seed, and seeds used for the production of T2plants. Transgenic T2 plants are used for the evaluation ofsalt-tolerance as described above. Transgenic T2 and T3 plants are usedin field trials for the evaluation of salt tolerance.

Methods

Cloning of the Arabidopsis Na⁺/H⁺ Antiport cDNA (AtNHX1)

The full-length cDNA of AtNHX1 was cloned by us from an Arabidopisisthaliana (Columbia) seedling cDNA library (38). The library wasinitially screened with an EST (GenBank # T75860; FIG. 8( h)) obtainedfrom the Arabidopsis Biological Resource Center (ABRC) that showedhomology to Arabidopsis genomic sequence (A-TM021 B04.4). The inventionincludes nucleic acid molecules of between about: 500-1000, 1000-15001500-1600, 1600-1700, 1700-2000 or 2000-2500 or greater than 2500nucleotides including the EST sequence (or a sequence having at leastabout: 35, 35, 55, 65, 75, 85, 90, 95, 99, 99.5 sequence identity to theEST sequence or the polypeptide encoded by the EST sequence) and whichencodes a polypeptide that extrudes monovalent (preferably potassiumions or lithium ions, most preferably sodium ions) out of the cytosolfor preparation of transgenic plants and host cells, and in the othermethods of the invention described below. These sequences are useful inthe methods of the invention described above (for example as a probe,research uses, hybridization). The Arabidopsis genomic sequencepredicted a polypeptide of 457 amino acids. Plaques that hybridized withthe labeled EST probe were subjected to a secondary screen using the PCRproduct from the nested amplification of a region coding for theN-terminal portion of the predicted polypeptide. The forward primer,based on the predicted start codon of the polypeptide (Primer-NT),5-GCCATGTTGGATTCTCTAGTGTCG-3 SEQ ID NO:11 and the reverse primer, basedon the stop codon predicted from the EST (Primer-CT),5′-CCGAATTCTCAAAGCTTTTCTTCCACG-3′ SEQ ID NO:12, were used to amplify a1.7 kb product from the seedling library. This product was purified byagarose gel electrophoresis and used as the template for a secondamplification using primer-NT and a reverse primer (primer-C) based onthe genomic sequence, 5′-CGGMTTCACAGAAAAACACAGTGAGGAT-3′ SEQ ID NO:13.The resulting 900 bp fragment served as the template for the probe usedin the secondary screen. The pure plaques obtained in the secondaryscreen were tested by PCR using the primer-NT, primer-CT combination.Three of the plaques, from which a 1.7 kb product was amplified, wereselected for excision of the phagemid. Single colonies containing theexcised phagemid were grown in liquid culture. Aliquots of each of thesecultures were used as templates for the PCR amplification of the regionbound by the library plasmid to the 5′ side of the clone (T3 promoter)and the reverse primer C. In one clone, a 1.2 kb fragment was amplified,which implied that the clone had an upstream untranslated region ofapproximately 300 bp. This clone was selected for complete sequencing.

Cloning of the Arabidopsis AtNHX3 Na+/H+ Antiport cDNA

The full-length AtNHX3 cDNA was cloned from an Arabidopsis thaliana(Columbia) seedling cDNA library. PCR primers were designed for theamplification of the AtNHX3 sequence based on a BAC DNA sequence (MTE17)with a predicted amino acid sequence showing homology to AtNHX1. Theforward primer (X6F), 5′-CCTCAGGTGATACCMTCTCA-3 SEQ ID NO:32 and thereverse primer (X6REV), 5-GATCCAATGTAACACCGGAG-3 SEQ ID NO:33 were usedto amplify a 1.2 kb product from the seedling library by PCR. Thisproduct was purified by agarose gel electrophoresis and used as a probein hybridization screening of the seedling cDNA library. Plaques thathybridized with the labeled probe were subjected to a secondary screenusing the 1.2 kb PCR product as a probe. Pure plaques obtained in thesecondary screen were tested by PCR using primer −X6F, primer −X6REVcombination. Only one of the plaques had the 1.2 kb product amplifiedfrom it. This plaque was used for excision of the phagemid. This clonewas used for complete sequencing.

Cloning of the Arabidopsis AtNHX5 and AtNHX4 Na+/H+ Antiport cDNAs

Full length AtNHX3 and AtNHX4 cDNAs were cloned by us from anArabidopsis thaliana (Columbia) seedling cDNA libraries (CD4-15 andCD4-16; Arabidopsis Stock Center, Columbus, Ohio). PCR primers weredesigned for the amplification of a genomic sequence based on a BAC DNAsequence (F20D21) with a predicted amino acid sequence showing homologyto both AtNHX1 and AtNHX2. The forward primer (NHX7F),5-TTCGTTCTCGGCCATGTCC-3 SEQ ID NO:34 and the reverse primer (NHX7REV),5-CGGAGAGACCAACACCTTCTGC-3 SEQ ID NO:35 were used to amplify a 2.2 kbproduct using Arabidopsis thaliana (Columbia) genomic DNA as a template.This product was purified by agarose gel electrophoresis and used as aprobe in hybridization screening of the seedling cDNA libraries. Plaquesthat hybridized with the labeled probe were subjected to a secondaryscreen using the 2.2 kb PCR product as a probe. Pure plaques were usedas templates for the PCR amplification of the region bound by thelibrary plasmid using the T3 and T7 promoter sequences as primers. Twoindependent clones (insert sizes of 1.7 kb and 2.1 kb) were selected forphagemid excision and complete sequencing.

Southern Blot Analysis

Genomic DNA was isolated from mature leaf tissue of Arabidopsis thaliana(Columbia). 10 ug of this genomic DNA was digested with ClaI, EcoR1,XbaI, or HindIII, fractionated on 0.7% agarose gel, and transferred toHybond N⁺ membrane (Amersham) according to manufacturers instructions.Overnight hybridization was performed at 65° C. in Amershamhybridization buffer with AtNHX1 cDNA fragments labeled with ³²P by therandom priming method. The final wash was in 0.1×SSPE, 0.1% SDS at 65°C. Hybridization signals were detected by autoradiography on BioMaxhyperfilm (Kodak).

Northern Blot Analysis

Arabidopsis thaliana ecotype Columbia was grown either on verticalplates on medium containing 0.5×MS salts and 1% agar at 20-25° C. undercontinuous fluorescent light for 1.5 weeks or in soil at 20-25° C. underfluorescent light and incandescent light with a 14 hour photo period for3-4 weeks. Total RNA was isolated from flower, leaf, and inflorescencestems of the mature plants and from root and shoot tissues of thevertically grown seedlings using TRIZOL reagent (GibcoBRL). 40 ug of RNAwas electrophoresed and transferred to Hybond N+membrane (Amersham)according to manufacturers instructions. Methylene blue was used tovisualize the 26S and 18S ribosomal RNA for quantitation. The blottedRNA was hybridized and washed as described for the southern blotanalysis.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. All such modifications are intended to beincluded within the scope of the appended claims.

Generation of Transgenic Arabidopsis Plants Overexpressing AtNHX1

An AtNHX1 PCR product was amplified using Vent DNA polymerase (NewEngland Biolabs) with the following primers (SE-ATX1-SalI5′-CGCGTCGACATGTTGGATTCTCTAGTGTCG-3′ SEQ ID NO:37 and ATXCT25′-CCGMTTCTCAAAGCTTTTCTTCCACG-3′ SEQ ID NO:12). This product wasdigested with SalI gel purified and used in a ligation reaction alongwith pBISNI prevously digested with SalI and SmaI and gel purified. Theresulting vector pBISNI-AtNHX1 contained the AtNHX1 open reading framein a sense orientation under the control of the super promoter.

Overexpression constructs carrying the AtNHX1 cDNA are introduced intoan electro-competent Agrobacterium tumefaciens (LBA4404) byelectroporation. The Agrobacteria are plated on LB plates containing 50mg/L kanamycin and grown for ˜2 days at 30° C. to select for bacteriacarrying the overexpression constructs. One liter liquid. LB+kanamycin(50 mg/L) is inoculated with a single Agrobacterium colony selected fromthe LB (kanamycin 50 mg/L) plates. The culture is grown to a minimum ofOD=1 (600 nm) for 2-3 days. The Agrobacteria are then pelleted andresuspended in 1 L infiltration medium (IM−0.5×MS salts; 0.5 g/L MES; 5%sucrose; 0.03% Silwet L-77). Flowering Arabidopsis plants with primarybolts reaching ˜15 cm are used for the transformation procedure (T1).Pots of Arabidopsis plants are dunked into the 1M solution containingthe Agrobacteria and left submerged for 2-6 minutes. The same procedurecan be repeated after 8-12 days on the same plants. Plants are allowedto senesce, the plants are dried thoroughly and the seeds harvested.Seeds are plated on agar plates containing 25 mg/L kanamycin in a mediumcontaining MS salts, 0.8% (w/v) agar adjusted to pH 6.0 with KOH. Theplates are transferred to a growth room at 25° C. under continuouslight. After 10 days the kanamycin-resistant seedlings (T2) aretransferred to small pots containing vermiculite. At senescence (˜8weeks) the seeds are collected from single plants and plated on agarplates containing MS salts and 25 mg/L kanamycin. After 10 days thekanamycin-resistant seedlings (T3) are transferred to small potscontaining vermiculite. Seeds produced by these plants are germinatedand used to assess salt tolerance of the transgenic plants.

Assessment of Salt Tolerance in Transgenic Plants

This procedure is described in the legend for FIG. 7.

The present invention has been described in detail and with particularreference to the preferred embodiments; however, it will be understoodby one having ordinary skill in the art that changes can be made theretowithout departing from the spirit and scope of the invention.

All articles, patents and other documents described in this application(including Genbank sequences and/or accession numbers), U.S. applicationSer. No. 60/078,474 (filed Mar. 18, 1998), U.S. application Ser. No.60/116,111 (filed Jan. 15, 1998) and U.S. Pat. Nos. 5,612,191,5,763,211, 5,750,848 and 5,681,714, are incorporated by reference intheir entirety to the same extent as if each individual publication,patent or document was specifically and individually indicated to beincorporated by reference in its entirety. They are also incorporated tothe extent that they supplement, explain, provide a background for, orteach methodology, techniques and/or compositions employed herein.

TABLE 4 Hybridization Wash High stringency (very similar sequences)55-65° C. 60-65° C. 5xSSC 0.1xSSC 2% SDS 0.1% SDS 100 μg/ml SSDNA Highstringency (similar sequences) (moderate stringency) 40-50° C. 50-50° C.5xSSC 0.1xSSC 2% SDS 0.1% SDS 100 μg/ml SSDNA Low stringency (lowsimilarity among sequences, i.e. many sequences similar) 30-40° C. 40-50° C. 5xSSC 2xSSC 2% SDS 0.2% SDS 100 μg/ml SSDNA Abbreviations: SSC =sodium chloride-sodium citrate buffer SSDNA = single stranded DNA

TABLE 5 List of Plants Alfalfa Almond Apple Apricot ArabidopsisArtichoke Atriplex Avocado Barley Beet Birch Brassica Cabbage CacaoCantaloup/cantalope Carnations Castorbean Caulifower Celery CloverCoffee Corn Cotton Cucumber Garlic Grape Grapefruit Hemp Hops LettuceMaple Melon Mustard Oak Oat Olive Onion Orange Pea Peach Pear PepperPine Plum Poplar Potato Prune Radish Rape Rice Roses Rye Sorghum SoybeanSpinach Squash Strawberries Sunflower Sweet corn Tobacco Tomato Wheat

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1. An isolated nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO: 3 or a nucleic acid that encodes SEQ NO:
 4. 2. A vectorcomprising the nucleic acid molecule of claim
 1. 3. The vector of claim2 comprising a promoter, wherein the promoter is operatively linked tothe nucleic acid molecule, wherein the promoter is selected from thegroup consisting of a super promoter, a 35S promoter of cauliflowermosaic virus, a drought-inducible promoter, an ABA-inducible promoter, aheat shock-inducible promoter, a salt-inducible promoter, acopper-inducible promoter, a steroid-inducible promoter and atissue-specific promoter.
 4. A host cell comprising thd vector of claim2.
 5. The host cell of claim 4, wherein said host cell is selected fromthe group consisting of a fungal cell, a yeast cell, a bacterial celland a plant cell.
 6. A transgenic plant, a plant part, a seed, a plantcell or progeny thereof, wherein the plant, plant part, a seed, plantcell or progeny comprises a construct comprising the nucleic acidmolecule of claim
 1. 7. The plant part of claim 6, comprising all orpart of a leaf, a flower, a stem, a root or a tuber.
 8. The plant, plantpart, seed, plant cell or progeny of claim 6, wherein the plant, plantpart, seed, plant cell or progeny is of a species selected from thegroup consisting of potato, tomato, Brassica, cotton, sunflower,strawberry, spinach, lettuce, rice, soybean, corn, wheat rye, barley,atriplex, sorghum, alfalfa, , Salicornia, almond, apple, apricot,Arabidopsis, artichoke, avocado, beet birch, cabbage, cacao,cantaloupe/cantaloup, carnation, castorbean, cauliflower, celery,clover, coffee, cucumber, garlic, grape, grape:fruit, hemp, hops, maple,melon, mustard, oak, oat, olive, onion, orange, pea, peach, pear,peanut, pepper, pine, plum, poplar, prune, radish, rape, rose, squash,sweet corn, and tobacco.
 9. The plant, plant part, seed, plant cell orprogeny of claim 6, wherein the plant is a dicot plant.
 10. The plant,plant part, seed, plant cell or progeny of claim 6, wherein the plant isa monocot plant.
 11. A tranagenic plant comprising recombinant DNA,wherein the recombinant DNA encodes a polypeptide comprising the peptidesequence of SEQ ID NO:
 4. 12. A transgenic plant comprising arecombinant nucleic acid molecule , wherein said nucleic acid moleculecomprises SEQ ID NO:3 or a nucleic acid encoding SEQ ID NO:
 4. 13. Thetransgenic plant of claim 12, wherein the recombinant nucleic acidmolecule is operatively linked to a constitutive promoter sequence or eninducible promoter sequence that provides transcription of therecombinant nucleic acid molecule in the plant.
 14. The transgenic plantof claim 12, wherein the recombinant nucleic acid molecule is chemicallysynthesized.
 15. The transgenic plant of claim 12, wherein therecombinant nucleic acid mdleeule is Isolated from Arabidopsis thaliana.16. An expression transgene comprising a recombinant nucleic acidmolecule operably linked to a promoter selected from the groupconsisting of a super promoter, a 35S promoter of cauliflower mosaicvirus, a drought-inducible promoter, an ABA-inducible promoter, a heatshock-inducible promoter, a salt-inducible promoter, a copper-induciblepromoter, a steroid-inducible promoter and a tissue-specific promoter,wherein said nucleic acid molecule comprises SEQ ID NO: 3 or a nucleicacid encoding SEQ ID NO:
 4. 17. A plant, a plant part, or a seed, aplant cell or progeny thereof, comprising the expression transgene ofclaim
 16. 18. The plant part of claim 17, comprising all or part of aleaf, a flower, a stem, a root or a tuber.
 19. The plant, plant part,seed, plant cell or progeny of claim 17, wherein the plant, plant part,seed1 plant cell or progeny is of a species selected from the groupconsisting of potato, tomato, Brassica, cotton, sunflower, strawberry,spinach, lettuce, rice, soybean, corn, wheat rye, barley, atriplex,sorghum, alfalfa, Salicornia, almond, apple, apricot, Arabidopsis,artichoke, avocado, beet, birch, cabbage, cacao, cantaloupe/cantaloup,carnation, castorbean, cauliflower, celery, clover, coffee, cucumber,garlic, grape, grapefruit, hemp, hops, maple, melon, mustard, oak, oat,olive, anion, orange, pea, peach, pear, peanut, pepper, pine, plum,poplar, prune, radish, rape, rose, squash, sweet corn, and tobacco. 20.The plant, plant part, seed, plant cell or progeny of claim 17, whereinthe plant is a dicot plant.
 21. The plant, plant part, seed, plant cellor progeny of claim 17, wherein the plant is a monocot plant.
 22. Amethod for producing a recombinant plant cell that expresses a nucleicacid molecule, wherein the method comprises introducing into a plantcell the expression transgene of claim
 16. 23. A method of producing agenetically transformed plant' wherein the method comprises regeneratinga genetically transformed plant from the plant cell, seed, plant part orprogeny thereof of claim
 17. 24. A transgenic plant produced accordingto the method of claim
 23. 25. A method for expressing a polypeptide ina plant cell, wherein the method comprises culturing the plant cell orprogeny of claim 17 under conditions suitable for gene expression.
 26. Amethod for producing a tranagenic plant comprising transforming a plantwith the expression transgene of claim
 16. 27. A method of producing ageneticaily transformed plant, wherein the method comprises: (a) cloningor synthesizing a nucleic acid molecule comprising SEQ ID NO: 3 or anucleic acid encoding SEQ ID NO: 4, (b) inserting the nucleic acidmolecule into a vector so that the nucleic acid molecule is operablylinked to a promoter; (c) inserting the vector into a plant cell orplant seed; and (d) degenerating a plant from the plant cell or plantseed.
 28. A transgenic plant produced according to the method of claim27.